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Image of a Plant cell and an Animal cell together

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Image of a Plant cell and an Animal cell together

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A double membrane layer that separates the contents of the nucleus from the rest of the cell.

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They are used for the creating of proteins through the translation process of tRNA interaction with mRNA within the ribosome.

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= dynamic organelles that can assemble, disassemble, or regulate their length in response to cellular and environmental cues

located at the surface of many cell types

Signaling from the cilia causes the autophagic machinery to trigger autophagosome formation

Can be either motile or nonmotile. Motile cilia is used to move the cell while nonmotile cilia/primary cilia are used to sense external and internal cues to the organism

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This membrane provides protection for a cell, transports nutrients into the cell, and sends toxic substances out.

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They are used for editing, collecting, and shipping products manufactured by the Endoplasmic Reticulum. - Paraphrased from the Textbook.

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Site for ATP production and respiration. they have their own genome but a large majority of the cell proteins are encoded by the nuclear genome. Its a critical central pathway for metabolism to occur.

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Cilia are basically flagella but to prevent confusion with bacterial flagella (which are structurally different from eukaryotic flagella) the term cilium is preferred over flagella.

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The Cisternae helps in packaging, modification, and transportation functions

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Used to break down materials using enzymes contented within.

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The three primary functions of the Golgi apparatus are the transport, sorting and modification of both protein and lipid, and the protein composition of the organelle reflects these functions.

https://www.ncbi.nlm.nih.gov/books/NBK6268/#:~:text=The%20three%20primary%20functions%20of,the%20organelle%20reflects%

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General Mechanisms and Pathways

In eukaryotes, different cellular functions are confined to specific membrane-bound organelles. Enzymes that mediate these functions are synthesized on cytosolic ribosomes (with the exception of proteins encoded by mitochondrial and chloroplast genomes) and thus need to be sorted and delivered from this site to the appropriate organelle. As originally described by Blobel, non-cytosolic proteins must contain a signal, or address label, that tells the cell where to put them. Other proteins (receptors) act as postmen reading the address labels by molecular recognition and delivering their protein cargo to their home organelle, or a "delivery truck" (transport vesicles or tubules) heading in the right direction. The Golgi apparatus is the sorting, packaging and distribution center of the exocytic pathway, handling proteins and lipids destined for the ER, plasma membrane, endosomes and lysosomes or the Golgi itself ( Fig. 5).

Membrane-bound vesicles, often wearing a proteinacious coat, mediate protein transport from the Golgi apparatus to other organelles.⁻ These vesicle coat proteins are thought to bend the membrane during vesicle budding, and also help to select and concentrate cargo proteins within the vesicles. Thus, the coat components often define the identity of these vesicles. A few types of coated vesicles generated from the Golgi apparatus have been well characterized that mediate different steps of protein transport. COPI-coated vesicles bud from all levels of the Golgi and are required for the retrograde transport of escaped ER residents back to the ER. These vesicles also appear to mediate protein transport between Golgi cisternae, although whether they mediate anterograde (forward) transport, retrograde transport, or both has been the subject of intense debate. Clathrin-coated vesicles (CCVs) form at the TGN, or immature secretory granules (ISG in Fig. 5), and carry proteins to endosomes. For example, lysosomal enzymes bearing the mannose-6-phosphate (M6P) determinant bind to the M6P receptor in the TGN and the complex is packaged into CCVs for initial delivery to an endosome. The lysosomal enzymes dissociate from the M6P receptor in the acidic environment of the endosome, allowing the receptor to recycle back to the TGN and the subsequent delivery of enzymes to the lysosome.

In addition, the TGN produces several different secretory vesicles with no known coat. Some cells will produce both "constitutive" vesicles (or tubules) that will fuse to the plasma membrane without the need for a stimulus, as well as "regulated" vesicles (or secretory granules) that require a calcium influx to drive fusion with the plasma membrane (Chapter 5). Moreover, polarized cells will segregate apical from basolateral proteins in the TGN and package these proteins into distinct vesicles. The basolateral proteins appear to be packaged into CCVs at the TGN and whether these vesicles are targeted directly to the basolateral membrane or initially to an endosome is unclear., Mechanisms for transport vesicle formation and targeting will be covered in greater detail in other chapters.

Golgi Protein Localization

Golgi resident proteins, such as the glycosyltransferases, are preferentially localized in different Golgi regions but the mechanism for localizing these enzymes to specific cisternae is unknown. All known Golgi glycosyltransferases are type II integral membrane proteins and localization signals have been found within the cytosolic tails, transmembrane domains (TMDs) and lumenal domains for different enzymes. No specific sequence comprising a Golgi localization signal that is shared by multiple proteins, such as the KDEL motif found on soluble ER proteins, has been identified thus far. Nor has any "receptor" been defined that recognizes a Golgi localization signal operating in the cis - trans cisterna. The situation is a little better for TGN resident proteins such as furin and Kex2p, where specific signals in the cytosolic tails mediate localization., These proteins appear to cycle to endosomes, and/or the plasma membrane as part of their normal trafficking itinerary. The cytosolic tail signals of these proteins are similar to endocytosis signals and operate in retrieval from endosomes back to the TGN. The rest of this discussion will focus on proteins localized in cis - trans cisternae.

For many years, it was assumed that Golgi cisternae were stable structures and that resident enzymes were statically retained within a cisterna. Models for Golgi protein localization that were popular in the 1990's reflected this bias. For example, the "oligomerization" or "kin recognition" hypothesis suggested that residents of a particular cisterna (kin) would form aggregates that were too large to enter into vesicles moving cargo in the anterograde direction, and thus these aggregates were retained in the Golgi cisterna in which they were formed., While there is evidence for interaction between Golgi proteins, they do not appear to form large oligomers in vivo. In addition, the Golgi apparatus appears to transport large oligomers, such as collagen or algal scales, through the stack fairly efficiently., Therefore, formation of large oligomers per se would not prevent movement through the Golgi, and the "kin recognition" hypothesis, at least as originally proposed, appears to be untenable.

A second "bilayer-thickness" hypothesis stemmed from the observation that the length of a TMD Golgi localization signal seemed more important than its amino acid sequence. Bretscher and Munro noted that Golgi enzymes tend to have shorter TMDs than plasma membrane proteins. They suggested that differences in membrane thickness across the Golgi stack, determined by differences in cholesterol content, caused Golgi enzymes to partition into membranes with an appropriate bilayer thickness to fit the length of their TMDs. This partitioning would prevent the lateral diffusion of Golgi enzymes into forming anterograde vesicles with thicker bilayers. However, studies in insect cells indicate that cholesterol is not a major determinant of Golgi protein localization, and whether or not bilayer thickness, controlled by another means, contributes to this process has not been experimentally tested. Moreover, it appears that bilayer thickness is determined primarily by the protein component rather than the lipid component of membranes. Therefore, the high concentration of Golgi enzymes likely determines the thickness of the bilayer in Golgi membranes, rather than cholesterol content, and perhaps this serves a mechanism to reinforce segregation of Golgi enzymes from non-Golgi membrane proteins. It should also be noted that the bilayer thickness model suggests a mechanism for how localization signals within the transmembrane segment function, but does not explain how localization signals in the cytosolic tails and lumenal domains of different Golgi proteins operate.

Studies on the localization of two different Golgi glycosyltransferases from yeast and one from mammalian cells suggested that these proteins were not significantly "retained" in their compartment of residence, but were actively retrieved from later Golgi compartments.⁻ These observations suggested a more dynamic mechanism for Golgi protein localization than previously considered, analogous to the KDEL-dependent retrieval of ER proteins from the Golgi apparatus back to the ER in COPI-coated transport vesicles. In fact, COPI-coated vesicles appear to mediate retrograde transport of Golgi enzymes back to the ER and from later to earlier Golgi cisternae.⁻ At least some Golgi proteins continuously cycle all the way back into the ER as part of their normal trafficking itinerary, and it has been argued that all Golgi proteins continuously transit through the ER.,⁻ Thus, while the mechanism for Golgi protein recognition (i.e., a sorting receptor) is not defined, it appears that retrograde transport plays an important role in Golgi protein localization. The growing realization that Golgi enzymes are not static residents of cisternae has impacted current views on how proteins move through the Golgi in the anterograde direction.

Protein Transport Through the Golgi Apparatus

The mechanism by which secretory cargo moves through the Golgi apparatus is unknown although two very different models have been proposed and hotly debated. The "stationary cisternae / vesicular transport" model suggests that each cisterna of the Golgi is a stable entity and secretory cargo is transported from one cisterna to the next in vesicles moving in the anterograde direction. Proteins enter the Golgi by fusion of VTCs with a preexisting cis-most cisterna and exit from the TGN by being packaged into larger secretory vesicles for delivery to the plasma membrane ( Fig. 5A). The alternative "cisternal maturation" (or cisternal progression) model suggests that the cis cisterna forms de novo by fusion of ER-derived membrane (VTCs) and progressively moves down the stack towards the trans face as though on a conveyer belt, maturing into medial and then trans cisternae along the way. The TGN then fragments into vesicles and is thus consumed ( Fig. 5B). Cisternae are thought to mature by exporting cis Golgi enzymes in transport vesicles (COPI-coated) to a younger cisterna forming in the rear, while acquiring later Golgi enzymes from older cisternae. The latter model suggests that the Golgi is an outgrowth of the ER and this is consistent with the effect of brefeldin A on the Golgi. This drug induces a collapse of early Golgi cisternae into the ER and later compartments with endosomes, but after the drug is removed, Golgi enzymes are exported from the ER and the stack is rebuilt., Remarkably, the entire organelle can be disassembled by brefeldin A and rebuilt within a few minutes after the drug is removed, indicating a tremendous plasticity for this organelle.

The history of the two models for protein transport through the Golgi is quite interesting. Electron microscopists studying the Golgi apparatus in the late 1950's and 1960's originally suggested that cisterna are produced on the "forming face", progress across the stack and are consumed into secretory vesicles at the "maturing face".,⁻ However, as various Golgi markers became better characterized, it was argued that cisternal progression couldn't adequately explain how resident proteins stay in the Golgi apparatus as secretory material quickly passes through. Nor did it explain how the Golgi residents could be concentrated in specific cisternae or the role of the numerous small vesicles surrounding the stack. Moreover, secretory cargo seemed to move quite efficiently between two different Golgi stacks in experimentally fused cells. Thus, in the 1980s several investigators suggested that a stable compartment model with secretory material passing from cisterna to cisterna in vesicles would better explain the available data.,,, This model was boosted by the reconstitution of vesicle-mediated protein transport between Golgi cisternae by the Rothman lab, which provided a tremendous advance in defining the molecular mechanisms of protein transport. This in vitro assay was designed to measure the movement of VSV G protein (secretory cargo) in transport vesicles from purified "donor" Golgi membranes deficient for GlcNAc transferase to "acceptor" Golgi membranes containing this enzyme but lacking VSV G. It led to the discovery of coatomer (COPI), the role of the small GTP-binding protein ARF in budding COPI vesicles, and the function of NSF and SNARE proteins in vesicle targeting and fusion.⁻

While the stationary cisternae/vesicular transport model enjoyed substantial popularity in the 1980's and most of the 1990's, it was not universally accepted. Morphologists studying secretion of scales from algae continued to make a particularly good case for cisternal progression. These carbohydrate rich structures are large enough to be visible in electron micrographs, and in many species the scales are significantly larger than the COPI vesicles surrounding the Golgi apparatus. A wave of scale secretion can be induced by deflagellating the algae, and the scales are observed to move across the Golgi stack without entering into small transport vesicles. This mode of transport does not appear to be unique to algae as other groups has made similar observations for the secretion of collagen from mammalian fibroblasts., Collagen is a 300 nm long, rod-shaped protein that forms large electron-dense aggregates within the Golgi apparatus. Its folding into a triple helical conformation within the ER requires an unusual hydroxyproline modification and the iron-dependent prolyl hydroxylase can be reversibly inhibited by iron chelators. Thus, cells treated with the chelator accumulate unfolded procollagen in the ER and a wave of collagen secretion can be induced by removing the chelator. In these experiments, collagen was observed to travel across the stack of Golgi cisternae without entering into smaller vesicles. However, this interpretation is partly based on the assumption that anterograde vesicles are constrained to a 50-60 nm diameter. In similar studies, the Rothman and Orci groups argued that a different protein aggregate in transit through the Golgi could be found in large "megavesicles" adjacent to cisternae. While these studies did not resolve the controversy, the pendulum of consensus view started swinging back toward the cisternal maturation model.⁻

The role of COPI-coated vesicles is another important distinguishing feature of these two models and the discovery that COPI vesicles mediate retrograde transport to the ER further swayed opinion towards the cisternal maturation model. In the stationary cisternae model, COPI vesicles are proposed to carry cargo in the forward (anterograde) direction between cisternae, whereas with the cisternal maturation model, these vesicles are proposed to carry resident Golgi enzymes in the retrograde direction. COPI was first implicated in retrograde protein transport from a genetic screen in yeast for mutants defective in Golgi to ER retrograde transport of a reporter protein bearing a "KKXX" ER retrieval signal. Mutant alleles for most of the COPI subunits were isolated in this screen, which demanded a functional secretory pathway for delivery of the KKXX-reporter protein to the plasma membrane. COPI mutants isolated in this and other unbiased screens, all exhibit a defect in retrograde transport but transport many proteins efficiently through the Golgi to the cell surface at the nonpermissive temperature. Some proteins are trapped in the ER of COPI mutants and this is thought to reflect a defect in recycling cargo receptors needed for packaging these proteins in COPII vesicles. The role of COPI in mediating Golgi to ER retrograde transport is now well established,, but remember that COPI was initially discovered using an in vitro assay that reconstituted vesicle-mediated transport between Golgi cisternae. However, this assay may reconstitute packaging of the GlcNAc transferase into COPI vesicles and the delivery of this modifying enzyme to Golgi cisternae containing VSV G., These findings suggest that COPI vesicles can also mediate retrograde transport of resident Golgi enzymes between Golgi cisternae.

Immunoelectron microscopy has also been used to probe the contents of peri-Golgi COPI vesicles with conflicting reports. One group reported finding significant levels of Golgi resident enzymes and the KDEL-receptor in these vesicles while VSV G was largely absent. Another group reported finding two populations of COPI vesicles, one containing retrograde cargo (the KDEL-receptor) and the other containing VSV-G. The latter observation led to a hybrid model postulating that cisternal maturation was a "slow track" through the Golgi apparatus while smaller cargo could speed through the stack using the COPI vesicle "fast track". However, a direct comparison of the rate of transport for collagen and VSV G suggests that these two proteins move synchronously through the stack. Discrepancies in reports of COPI vesicle content and function may be explained by the growing evidence for discrete subpopulations of these vesicles. The Golgi region contains a number of large, coiled coil protein complexes called golgins that can tether vesicles to Golgi cisternae and perhaps control movement of vesicles across the stack. The CASP/golgin-84 complex can specifically bind to COPI vesicles containing Golgi enzymes but lacking ER retrograde or anterograde markers, whereas the p115-golgin tether can select COPI vesicles containing an anterograde cargo but lacking Golgi enzymes. Thus, it appears that not all COPI vesicles are created equal and perhaps each distinct transport step between Golgi cisternae uses a specific subpopulation of COPI vesicles.

However, the prevailing view that COPI vesicles are the major mediator of protein flux through the Golgi (anterograde or retrograde) may be inaccurate, and therefore the premise of using COPI vesicle content to distinguish competing models may not rest on a firm foundation. Yeast genetic studies indicate that anterograde transport of secretory cargo through the Golgi apparatus is efficient in the absence of COPI function,, while inactivation of Sec18 (NSF), and thereby SNARE function, immediately blocks anterograde movement of cargo within the Golgi., This suggests that anterograde transport requires multiple membrane fusion events that are independent of a COPI vesicle intermediate. Some Golgi proteins are mislocalized to a downstream compartment (the vacuole) in the COPI mutants,, notably those that cycle back to the ER, although the Golgi retains sufficient enzyme content to terminally glycosylate secretory cargo. If COPI is the sole mediator of retrograde transport of Golgi enzymes during cisternal maturation, we would expect a wholesale loss of the Golgi enzymes after inactivating COPI, which does not seem to occur. Moreover, there is good evidence for COPI-independent retrograde transport of Golgi enzymes to the ER in mammalian cells via a mechanism requiring Rab6 but no known vesicle coat protein. What mediates protein flux through this organelle if not COPI? One possibility is that transient tubular connections between unlike cisternae provide a conduit for the flow of Golgi enzymes in the retrograde direction to drive cisternal maturation, or anterograde flow of cargo through stable cisternae., These tubular connections would presumably require the SNARE machinery to form without a need for coat proteins, but how the directionality of protein flow would be controlled by this mechanism is unclear. Another possibility is that transport is mediated by an undefined vesicular intermediate that does not require COPI for formation.

Perhaps the most direct test of the cisternal maturation model is to visualize individual cisternae in living cells over time to determine if the content of resident enzymes changes or stays the same. Because of their close proximity to each other, individual cisternae of the mammalian Golgi cannot be distinguished by light microscopy, but the scattered cisternae of Saccharomyces cerevisiae are ideal for this type of analysis. Two different groups have used GFP and RFP fused to different Golgi proteins (markers of cis, medial and trans compartments) to monitor the residence time of these markers in individual cisterna., By the stationary cisternae model, one would expect a relatively long residence time for these proteins within their cisternae. Instead, cis-Golgi cisternae marked with a GFP fusion protein rapidly lost their green color while they acquired the red color of a trans-Golgi protein fused to RFP. Importantly, the rate of this color change was very similar to the rate of anterograde cargo transport through the Golgi. In addition, the color changes were always unidirectional in the cis to trans direction; a trans cisterna never acquired cis-Golgi enzymes. These data are inconsistent with a stationary cisternae model and strongly support a cisternal maturation model. Interestingly, Golgi cisternae matured in a COPI mutant although the rate of maturation was slower relative to a wild-type cell.

The majority of evidence in the literature now favors the cisternal maturation model and most investigators in the field have returned to this original view of a dynamic organelle in constant flux.,, However, some observations are difficult to reconcile with cisternal maturation. For example, incubation of mammalian cells at 20°C blocks exit of secreted proteins from the TGN while transport from the ER to the Golgi and through the stack is not inhibited. By the cisternal maturation model, one might anticipate that the 20°C block would cause an increase in the number of Golgi cisterna, but this does not occur. Instead cargo accumulates in bulging domains in the last few cisternae. To be consistent with a cisternal maturation mechanism, the TGN cisternae that are not consumed by fragmentation at 20C would have to join together by a homotypic fusion mechanism, which would suggest the number of cisternae in the cell is somehow tightly regulated.

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Secretion of proteins from eukaryotic cells requires the coordinated function of multiple organelles and cellular machineries. After synthesis and translocation into the endoplasmic reticulum, proteins are exported to the Golgi apparatus, a multi-compartment organelle that is the protein modifying, packaging and distribution center of the secretory pathway.

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A ribosome is an intercellular structure made of both RNA and protein, and it is the site of protein synthesis in the cell. The ribosome reads the messenger RNA (mRNA) sequence and translates that genetic code into a specified string of amino acids, which grow into long chains that fold to form proteins.

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A ribosome is an intercellular structure made of both RNA and protein, and it is the site of protein synthesis in the cell. The ribosome reads the messenger RNA (mRNA) sequence and translates that genetic code into a specified string of amino acids, which grow into long chains that fold to form proteins.

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A ribosome is an intercellular structure made of both RNA and protein, and it is the site of protein synthesis in the cell. The ribosome reads the messenger RNA (mRNA) sequence and translates that genetic code into a specified string of amino acids, which grow into long chains that fold to form proteins.

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Definition

A ribosome is an intercellular structure made of both RNA and protein, and it is the site of protein synthesis in the cell. The ribosome reads the messenger RNA (mRNA) sequence and translates that genetic code into a specified string of amino acids, which grow into long chains that fold to form proteins.

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Definition A ribosome is an intercellular structure made of both RNA and protein, and it is the site of protein synthesis in the cell. The ribosome reads the messenger RNA (mRNA) sequence and translates that genetic code into a specified string of amino acids, which grow into long chains that fold to form proteins.

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They are made of two parts each are rRNA.

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The Golgi Apparatus Structure and Function | The Endomembrane System

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It does calcium storage, protein synthesis, and lipid metabolism

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may be used to destroy invading viruses and bacteria

If a cell is damaged beyond repair, lysosomes can help it to self-destruct via “programmed cell death,” or apoptosis

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The endoplasmic reticulum changes shape in response to cellular cues, cell type, cell cycle state, and during the time the organism is growing.

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Cellular and Molecular Life Sciences

Abstract

The endoplasmic reticulum (ER) is a large, dynamic structure that serves many roles in the cell including calcium storage, protein synthesis and lipid metabolism. The diverse functions of the ER are performed by distinct domains; consisting of tubules, sheets and the nuclear envelope. Several proteins that contribute to the overall architecture and dynamics of the ER have been identified, but many questions remain as to how the ER changes shape in response to cellular cues, cell type, cell cycle state and during development of the organism. Here we discuss what is known about the dynamics of the ER, what questions remain, and how coordinated responses add to the layers of regulation in this dynamic organelle.

Keywords: Interphase, Mitosis, Unfolded protein response, Organization, Fertilization, Phosphorylation

Introduction

The ER is the largest organelle in the cell and is a major site of protein synthesis and transport, protein folding, lipid and steroid synthesis, carbohydrate metabolism and calcium storage [ 1- 7]. The multi-functional nature of this organelle requires a myriad of proteins, unique physical structures and coordination with and response to changes in the intracellular environment. Work from a variety of systems has revealed that the ER is composed of multiple different structural domains, each of which is associated with a specific function or functions. However, it is not yet clear how these functional subdomains are organized and how different functional domains translate into different structures.

Protein synthesis and folding

One of the major functions of the ER is to serve as a site for protein synthesis for secreted and integral membrane proteins [ 8], as well as a subpopulation of cytosolic proteins [ 1]. Protein synthesis requires localization of ribosomes to the cytosolic face of the ER, and the canonical pathway that regulates protein synthesis involves co-translational docking of the mRNA:ribosome complex on the ER membrane. Translation of secretory or integral membrane proteins initiates in the cytosol, then ribosomes containing these mRNAs are recruited to the ER membrane via a signal sequence within the amino terminus of the nascent polypeptide that is recognized and bound by the signal recognition particle (SRP) [ 9, 10]. The complex of mRNA:ribosome:nascent polypeptide:SRP is targeted to the ER where it docks on the SRP receptor [ 11, 12]. Translation continues on the ER and the emerging polypeptide can co-translationally enter the ER through the translocon [ 2], which is a channel that contains several Sec proteins and spans the lipid bilayer [ 13].

Also during this time, or in some cases once translation is complete [ 3], a signal peptidase cleaves the short signal peptide allowing the free protein to enter the ER lumen [ 14]. If the protein is destined to be an integral membrane protein, determined by the presence of a stretch of hydrophobic residues or stop-transfer membrane anchor sequence, translocation will pause [ 15]. At this point the protein will be shifted laterally and become anchored within the phospholipid bilayer where it remains [ 15]. Transmembrane proteins can either contain one hydrophobic stretch of amino acids, and are classified as single pass transmembrane proteins, or contain multiple regions that cross the membrane and are classified as multi-pass transmembrane proteins [ 3].

If the protein is not destined to be integrated into the membrane, but instead enter the secretory pathway or the lumen of membrane-bound organelles, the protein begins the process of transport. Once translation is complete and the signal peptide has been cleaved the ribosomes are released back into the cytosol [ 16, 17]. For mRNAs translated by stably-bound ER ribosomes, mRNAs are released and ribosomes may remain bound to the ER and participate in multiple rounds of translation [ 18, 19]. For cytosolic proteins translated on ER-bound ribosomes it is not clear how these mRNAs are recruited to the ER or what populations of ribosomes are utilized to initiate translation, although a recent study indicates that the ER-resident protein p180 may play a role in the translation-independent recruitment of mRNAs to the ER [ 20].

Following protein synthesis and translocation into the ER lumen, a protein destined for secretion must undergo proper folding and modifications, with the aid of chaperones and folding enzymes. These modifications include N-linked glycosylation, disulfide bond formation and oligomerization [ 3]. At this point the fate of the secretory proteins is determined. If the protein functions in the ER, for example as a chaperone, then proper folding will commence. If the protein is destined for secretion, it will be released by the chaperones and packaged for travel through the Golgi on to a final destination (such as the plasma membrane or secreted) or move into peroxisomes [ 21]. Additionally, the cytosolic regions of the transmembrane protein may interact with cytosolic proteins or chaperones to properly fold these domains.

On the other hand, even with several proteins and complexes dedicated to folding proteins properly, a fraction of proteins do not achieve native and functional form and are either misfolded or aggregated [ 22]. These proteins can either remain in the ER or enter the ER-associated degradation (ERAD) pathway mediated by the proteasome, assuring that aberrant polypeptides do not inadvertently enter the secretory pathway [ 23]. Recognition of misfolded proteins, followed by clearing of these aggregates through the ERAD pathway, needs to be tightly controlled so as not to affect cellular function [ 23]. Interestingly, there are several connections to activation of ER stress response pathways and pathological human conditions. Several neurodegenerative protein misfolding diseases, such as Alzheimer's disease, activate ER stress response pathways. Additionally, activation of the ER stress response pathway is observed in diabetes, inflammatory bowel disease, and various cancers. How ER stress response pathways play a role in these pathologies is an active area of research and various components of the stress response pathways are being investigated as potential therapeutic targets [ 24]. In general, the protein synthesis functions of the ER are confined to ER sheets and regulation of ER structure by RNA localization and ER stress will be covered later in this review.

Lipid biogenesis

While the ER is a major site of protein synthesis, it is also a site of bulk membrane lipid biogenesis [ 4], which occurs in the endomembrane compartment that includes the ER and Golgi apparatus. Proteins and phospholipids, which are the major lipid component of membranes, are transferred and biochemically modified in the region of the ER that is in close juxtaposition to the Golgi apparatus [ 25]. This region, known as the ER-Golgi intermediate compartment (ERGIC), is rich in tubules and vesicles [ 4]. Once lipids are mobilized to the ERGIC they are distributed throughout the cell through organelle contacts or secretory vesicles [ 26]. The cis-Golgi, which is the closest structure to the ERGIC, leads to the trans-Golgi network where vesicles carrying newly synthesized secretory proteins from the ER form and bud [ 4]. The trans-Golgi network has traditionally been viewed as the main sorting station in the cell where cytosolic cargo adaptors are recruited to bind, indirectly or directly, and transport proteins or lipids [ 27].

Calcium (Ca²⁺) metabolism

Finally, while the ER is a major site of synthesis and transport of a variety of biomolecules, it is also a major store of intracellular Ca ²⁺ [ 28, 29]. The typical cytosolic concentration of Ca ²⁺ is ~100 nM, while the Ca ²⁺ concentration in the lumen of the ER is 100-800 μM, and the extracellular Ca ²⁺ concentration is ~2 mM [ 6, 30]. The ER contains several calcium channels, ryanodine receptors and inositol 1,4,5-trisphosphate (IP ₃) receptors (IP ₃R) that are responsible for releasing Ca ²⁺ from the ER into the cytosol when intracellular levels are low [ 6]. Ca ²⁺ release occurs when phospholipase C (PLC) is stimulated through G protein-coupled receptor (GPCR) activation [ 31] and cleaves phosphatidylinositol 4,5 bisphosphate (PIP ₂) into diacyl-glycerol (DAG) and IP ₃, which can then bind the IP ₃R leading to Ca ²⁺ release and transient increase in intracellular Ca ²⁺ levels [ 6]. Ryanodine receptors (RyRs) act through Ca ²⁺-induced Ca ²⁺ release (CICR), when the receptors bind Ca ²⁺ in response to increased cytoplasmic levels of Ca ²⁺ [ 32]. In addition, depolarization of t-tubule membranes can lead to conformational changes in voltage-dependent Ca ²⁺ channels, such as dyhydropyridine receptors (DHPRs), which interact and activate RyRs leading to Ca ²⁺ release [ 33]. Furthermore, Ca ²⁺ can leak from the ER into the cytoplasm only to be pumped back into the ER via sarcoendoplasmic reticular Ca ²⁺ ATPases (SERCAs), or can enter the cell from the extracellular media, adding to the layers of regulation [ 6]. If ER stores of Ca ²⁺ are rapidly depleted through IP ₃ receptor (IP ₃R)-mediated release a mechanism for Ca ²⁺ entry into the cell is activated, known as store-operated Ca ²⁺ entry (SOCE) [ 6, 34]. After ER luminal Ca ²⁺ depletion, STIM1 proteins cluster in regions of ER abutting the plasma membrane. At these regions, clustered STIM1 traps plasma membrane-diffusing Orai1 subunits [ 35, 36] and assembles them into active Ca ²⁺ release-activated channels (CRAC) allowing for uptake of extracellular Ca ²⁺ into the ER lumen to restore Ca ²⁺ levels [ 37- 39]. Interestingly, SOCE and activation of CRAC does not depend on, nor sense, changes in levels of Ca ²⁺ in the cytoplasm [ 6], but senses and responds to changes in luminal Ca ²⁺ concentration.

Calcium is a widespread signaling molecule that can affect diverse processes including localization, function and association of proteins, either with other proteins, organelles or nucleic acids. Release of Ca ²⁺ can result in a wave of Ca ²⁺ that moves through the entire cell [ 40], a gradient of Ca ²⁺ from the source of release, or a spatially-restricted wave from clustered channels known as a Ca ²⁺ spark [ 41]. One of the most well-studied Ca ²⁺ release events occurs at fertilization following sperm entry [ 40, 42], but also occurs during muscle contraction and secretion [ 6] as well as neuronal processes including neurotransmitter release [ 43]. We will highlight recent evidence that Ca ²⁺ may also play a role in reshaping the ER in response to cellular signals.

Regulation of ER shape and function

The ER is a complex organelle, involved in protein and lipid synthesis, calcium regulation and interactions with other organelles. The complexity of the ER is reflected in an equally complex physical architecture. The ER is composed of a continuous membrane system that includes the nuclear envelope (NE) and the peripheral ER, defined by flat sheets and branched tubules (Fig. 1 ). The shape and distribution of these ER domains is regulated by a variety of integral membrane proteins and interactions with other organelles and the cytoskeleton. These interactions are dynamic in nature and reflect changes within the cell, either through cell cycle or developmental state, cell differentiation, intracellular signals or protein interactions. While it is generally known how the basic shapes of ER sheets and tubules are determined, it is relatively unclear how changes in shape or the ratio of sheets to tubules occur in response to specific cellular signals.

Here, we will discuss what is known about how the structures of ER are formed, how the dynamics of the ER are regulated, and how these dynamics change in response to cell cycle state and cellular cues. In addition, we provide examples of how the proteins that are involved in contributing to ER shape are influenced by these cellular cues, such as calcium release, and how this is reflected in the dynamics of the ER and ultimately the function of specialized cells that display varying ratios of sheets to tubules.

ER structure

There have been several excellent, recent reviews that cover the topic of general ER structure in detail [ 7, 44- 48], so we will limit our review of the basic ER structure to only those factors that may play a role in changing the shape of ER in response to signaling. The ER consists of the nuclear envelope and the peripheral ER, which includes smooth tubules and rough sheets. While the ER is defined as an interconnected network with a continuous membrane, the different structures that make up the ER perform very diverse and specialized functions within the cell.

The nuclear envelope is made up of two lipid bilayers, the inner nuclear membrane (INM) and outer nuclear membrane (ONM), and shares a common lumen with the peripheral ER. Hundreds of nuclear pores spanning the ONM and INM of the nuclear envelope allow transport of molecules, including RNAs and proteins, at various rates of diffusion or regulated transport depending on the size of the molecule. The nuclear envelope is connected to sheets, or cisternae, that make up part of the peripheral ER. Sheets are flat in nature consisting of two lipid bilayers with an intervening lumen, with curved regions located only at the membrane edges. Peripheral ER Sheets may vary in size, but the luminal spacing is very consistent, usually about 50 nm in mammals and 30 nm in yeast [ 49] (Fig. 2 ). Sheets are usually observed in a stacked conformation and are connected via regions of twisted membranes with helical edges [ 50]. These rough sheets, as defined by the high density of ribosomes on the cytosolic surface [ 51, 52], are the main site of synthesis, folding and post-translational modifications for secreted or membrane-bound proteins. In turn, far fewer ribosomes are present on the membrane surface of ER tubules [ 52], which is highly curved and smooth and may not accommodate the binding of large polysomes (Fig. 2 ). The tubular network is dynamic, continually rearranging and growing, and is defined by three-way junctions that connect individual tubules (Fig. 1 ). While tubules and sheets possess very different structural features, and hence play a role in different cellular processes, the luminal spacing of both tubules and sheets is similar [ 49, 52].

Interestingly, ER tubules and sheets are found in all eukaryotic cells [ 53], though the ratio of sheets to tubules varies in different cell types and reflects the different functions of these cells. For example, the ER architecture of specialized cells that synthesize vast amounts of secreted proteins, such as pancreatic secretory cells and B cells, is largely made up of sheets (Fig. 1 ). In turn, cells that are involved in processes including lipid synthesis, calcium signaling and sites of contact for other organelles possess an ER composed of primarily tubules (Fig. 1 ). Adrenal, liver and muscle cells are all examples of specialized cells with a predominantly tubular network and reflects the function of these cells [ 54].

An additional configuration of the peripheral ER includes cortical ER, which abuts the plasma membrane and displays an intermediate phenotype between sheets and tubules with membranes that are both highly curved as well as regions that are flat in nature. Calcium signaling occurs at the contact sites between the plasma membrane and the abutting cortical ER and is necessary for muscle contraction [ 55, 56]. Therefore, the morphology and intracellular location of the ER subdomains contribute to the function of these structures and hence the role of the specialized cell in which they are located.

Improved microscopy techniques have allowed for the characterization of different ER structures, and the ratios of these structures to one another, in specialized cell types. When comparing the roles of these cells in the organism, it is clear that the type and amount of peripheral ER present reflects the function of that particular cell type. It is still unclear how these ratios are generated and what cellular signaling pathways play a role in designating which ER type will be most prominent in a particular cell type.

ER shaping proteins

ER tubules

Peripheral ER structures are just as distinct and diverse as the set of proteins that contribute to their shape. Several proteins have been identified that promote specific ER structures, but perhaps the most well-studied group of proteins include the reticulon family of proteins that localize to tubules and the highly curved edges of ER sheets [ 51, 57]. These integral membrane proteins contribute to the bending of the membrane by forming a transmembrane hairpin topology that acts as a wedge, displacing lipids in the outer leaflet of the bilayer leading to curvature of the membranes [ 57]. These proteins tend to form oligomers and are much less mobile than other ER-resident proteins [ 58]. Overexpression of some reticulon isoforms leads to formation of long ER tubules at the expense of sheets [ 58]. In turn, depletion of reticulons, and hence the ability to bend membranes, leads to a reduction in the number of ER tubules, leading to an expansion of peripheral sheets [ 57, 59, 60]. Therefore, the level of reticulons within a cell determines the abundance and fine structure of ER tubules.

Reticulons do not act alone in shaping ER tubules. Members of the DP1/Yop1/REEP5/6 and REEP1-4 family, which are abundant ER-resident proteins that specifically localize to tubules and edges of sheets, also act as tubule-promoting factors. DP1/Yop1, or REEP5/6 [ 61], proteins share a similar transmembrane hairpin architecture with the reticulons (Fig. 2 ), leading to the stabilization of the curved membranes of tubules [ 57, 58, 62]. Interestingly, REEP1-4 proteins have a topology distinct from REEP5/6 suggesting that these proteins may have slightly different functions in shaping the ER than the closely related REEP5/6 proteins [ 63] (Fig. 2 ). Additionally, purified reticulons and DP1/Yop1 family proteins were able to induce tubule formation from purified vesicles [ 62], demonstrating that these proteins play an essential role in ER tubule growth.

Reticulons and DP1/Yop1 promote tubule formation, but additional factors are required to promote the formation of the tubular network and characteristic three-way junctions through homotypic fusion. Atlastins, members of the dynamin-like GTPase family, mediate these homotypic fusion events. Depletion by RNAi or expression of dominant-negative atlastin in cells results in a lack of fusion events leading to an abundance of long, unbranched tubules [ 61]. When a dominant-negative cytoplasmic fragment from Xenopus, which contains the GTPase domain but lacks the transmembrane domain and cytoplasmic tail [ 64], are introduced into Xenopus interphase extracts ER network formation was blocked [ 65]. Comparable point mutations that prevent dimerization of the cytoplasmic fragment of human atlastin [ 66] were made in the Xenopus cytoplasmic atlastin protein, added into interphase extract and had no effect on ER network formation [ 65]. Furthermore, antibodies directed against atlastin inhibit ER network formation when introduced into Xenopus egg extracts [ 61]. In Drosophila, atlastin depletion leads to ER fragmentation and purified atlastin is sufficient to catalyze GTP-dependent fusion of proteoliposomes [ 64, 66, 67]. Therefore, studies from multiple organisms, extracts and purified components indicate that atlastin is likely required for catalyzing homotypic vesicle fusion between ER membranes, which is important for proper network formation.

Recently, a few new key players have been identified that are involved in ER dynamics. Work using purified ER vesicles derived from Xenopus eggs has demonstrated that GTP is required for homotypic ER vesicle fusion in the absence of cytosolic factors [ 57, 68]. Previous studies indicated that GTPases are required for ER fusion events [ 69, 70], and a recent study utilized a proteomics approach to identify Rab10 as a factor required for ER assembly [ 71]. Knock-down of Rab10, or overexpression of a GDP-locked dominant-negative point mutant, in cultured human cells caused an increase in ER sheets and a decrease in tubules [ 71]. ER-ER fusion events occurred at regions where Rab10 was enriched. Rab10 was found to co-localize with several lipid-synthesizing enzymes, including phosphoinositol synthase (IS) and choline/ethanolamine phosphotransferase (CEPT1) [ 71], leading to the possibility that this may represent a previously unidentified ER subdomain or compartment. It is currently not clear what role Rab10 plays in the ER vesicle fusion reaction or how homotypic ER vesicle fusions are coupled to lipid synthesis.

Recent work has also identified a role for Rab18, which is targeted to the ER by Rab3 GTPase activating protein (GAP) complex, in ER dynamics. Depletion of Rab18 leads to a phenotype similar to that observed following Rab10 inhibition [ 72]. Additionally, when Rab10 is depleted, Rab18 redistributes to peripheral sheets [ 72]. Therefore, it appears that depletion of either Rab10 or Rab18 prevents the stabilization of ER tubule fusion, reducing the density of tubules resulting in an increase in ER sheets. Depletion of the Caenorhabditis elegans RAB-5, which has been previously implicated in early endosome function [ 73], phenocopies the peripheral ER defects seen in the RET-1 and YOP-1 (homologs of Rtn4a and DP1) depletions [ 70]. In addition to the role RAB-5 plays in peripheral ER formation, kinetics of nuclear envelope disassembly is affected in these mutants [ 70].

In addition to GTPases that may play a direct role in homotypic membrane fusion of vesicles, recent work has demonstrated a role for lipid synthesizing enzymes in controlling the shape and organization of the ER. Inhibition of C-terminal domain (CTD) nuclear envelope phosphatase-1 (CNEP-1), which is enriched on the nuclear envelope and promotes the synthesis of membrane phospholipids, led to the appearance of ectopic sheets that encased the nuclear envelope, interfering with nuclear envelope breakdown [ 74]. These results reflect the interconnected network of proteins and functions that play a role in shaping the structures of the ER.

The ER is a very dynamic network that is constantly undergoing rearrangements and remodeling [ 75]. ER tubules are continually fusing and branching resulting in the creation of new three-way junctions. In a competing process, junction sliding and tubule ring closure leads to loss of three-way junctions and the characteristic polygonal structure [ 76]. Very little is known about the complexes controlling this process, but it was recently discovered that Lunapark (Lnp1) localizes to and stabilizes three-way junctions [ 77, 78]. Lnp1 binds to reticulons and Yop1, and localization of Lnp1 to junctions is regulated by Sey1p, the yeast homolog of atlastin [ 78]. Loss of Lnp1 leads to a collapsed and densely reticulated ER network in yeast and human cultured cells [ 77, 78], though only half of the junctions are bound to Lnp1 [ 77], which reflects the fluidity of the ER network. If Lnp1 is overexpressed, the protein localizes to the peripheral ER and induces the formation of a large polygonal tubular network [ 79]. Additionally, formation of this network was inhibited by Lnp1 mutations that blocked N-myristoylation [ 79], an attachment of myristic acid (a 14-carbon saturated fatty acid), indicating that this modification plays a critical role in Lnp1-induced effects on ER morphology. N-myristoylation is not required for membrane translocation, topology formation, or protein localization to the ER but may play a role in protein-protein or protein-lipid interactions that are required for morphological changes in the ER, though the exact molecular mechanism of action remains to be elucidated [ 79].

The actual mechanism for Lnp1-mediated stabilization of three-way junctions is unknown, though recent studies and insights from the structure and domains within the protein shed light on how Lnp1 stabilizes junctions [ 77, 78]. First, Lnp1 contains two transmembrane domains as well as a zinc finger domain, which is located on the cytoplasmic face of the ER membrane [ 77]. When cysteines were mutated within the zinc finger domain, the polygons became smaller and regions lacking cortical ER were more apparent as the number of cysteines mutated increased [ 78]. Therefore, mutations in the zinc finger domain may affect protein-protein interactions, complex formation or interfere with the distribution of resident lipids on the cytoplasmic face of the membrane causing deleterious effects on junction stabilization. In addition, the transmembrane domains may be acting as an inverted wedge, adding to the local negative curvature characteristic of three-way junctions [ 77], and acting opposite to the positive curvature promoted by reticulons. Another possibility is that multiple Lnp1 proteins may also act cooperatively together to stabilize the junction, or Lnp1 may be acting transiently to stabilize or modify lipids or other proteins at junctions [ 77].

In addition to proteins that regulate membrane structure and dynamics, there is accumulating evidence that changing the nucleic acid content of the ER can also impact ER shape. Early experiments showed that brief treatment of tissue culture cells with the translation inhibitor puromycin, which dissociates mRNA:ribosome complexes, leads to loss of ribosomes from the ER and a loss of ER sheets [ 51, 80]. This suggests that the presence of mRNA:ribosome complexes may stabilize ER sheets. In support of this hypothesis, our recent work identified an ER-localized ribonuclease, XendoU [ 81], that changes the RNA content of the ER in response to changes in free Ca ²⁺ concentration [ 82, 83]. These changes occur at physiologically relevant levels of ~1.5 μM, which mimics release of Ca ²⁺ from intra- and extracellular stores at fertilization [ 42, 84]. A subpopulation of XendoU localizes to the ER and co-immunoprecipitates with a number of ER-resident proteins [ 82]. Depletion of XendoU leads to the formation of long, unbranched tubules in Xenopus leavis egg extract, and rescue of this phenotype requires intact catalytic activity of the protein, indicating that the nuclease function is critical to proper ER network formation [ 82]. Furthermore, antibody addition to purified vesicles leads to a block in network formation, demonstrating that XendoU acts on the surface of ER membranes to regulate ER structure [ 82]. Interestingly, addition of 5′5′-dibromo BAPTA, a strong calcium chelator, blocked vesicle fusion in this system [ 68]. Depletion of XendoU also leads to a delay in replication and nuclear envelope closure [ 82], and BAPTA blocks nuclear envelope formation in Xenopus egg extract reconstitution experiments [ 85]. Together these results suggested that XendoU acts on membranes to degrade RNAs.

Upon vesicle fusion it was found that RNAs were degraded and released from the surface of membranes, suggesting that XendoU acts to degrade these RNAs, as well as release proteins, to clear patches of membrane to allow for vesicle formation leading to network formation [ 82]. Interestingly, when purified vesicles were treated with increasing concentrations of RNaseA and subjected to the same assay, an increasingly aberrant network formed with large vesicles that were unable to fuse [ 82]. Results from in vitro studies indicate that XendoU is activated on membranes in coordination with calcium release to locally degrade RNAs and clear patches of membranes leading to fusion in a controlled manner to fine tune network formation.

Lastly, similar to other proteins that play a role in tubule formation, knock-down of the human homolog EndoU in cultured human cells leads to an expansion of sheets [ 82]. Additionally, rescue of the expanded sheet phenotype depended on intact catalytic function as observed with recombinant protein in the extract system. Therefore, XendoU is an example of a protein that is activated in response to cellular cues to regulate proper ER formation, and further studies may reveal additional proteins that are regulated in this manner to fine tune organelle structure.

ER sheets

We have considered how tubules are formed and maintained, which leads the discussion to sheets, the other peripheral ER structure. First, we must consider how sheets are formed. Several mechanisms have been proposed, including the idea that integral membrane proteins can span the intraluminal space and form bridges, connecting the lipid bilayers [ 51, 86, 87]. These proteins may either stabilize the structure or define the distance between the two lipid layers based on the size of the proteins. Additionally, these proteins or protein complexes may form a scaffold that aids in the stabilization of the sheets or bring the two lipid membranes in closer proximity [ 86]. Several proteins including Climp63, p180 and kinectin have been implicated in the generation, maintenance and stabilization of ER sheets [ 51].

In addition to highly enriched membrane proteins and core components of the translocon, Climp63, a coiled-coiled protein with a single transmembrane domain, was identified along with kinectin and p180 in a mass spectrometry screen for abundant integral ER membrane proteins [ 51]. Through various techniques and in various cell types Climp63 was shown to be a highly abundant protein [ 88- 90] that localizes to perinuclear ER and is absent from the nuclear envelope [ 91, 92]. Very stable oligomers of Climp63 can form, restricting mobility of the protein along the membrane, promoting localization to the rough ER [ 92]. Overexpression of Climp63 leads to a massive proliferation of ER sheets while reduction in expression surprisingly does not lead to loss of sheets but instead a decrease in the distance between sheets [ 51]. Moreover, these sheets are spread diffusely throughout the cytoplasm, reminiscent of the phenotype of cells treated with the translation inhibitor puromycin [ 51]. This is interesting as the core components of the translocon, the protein channel that interacts with ribosomes and is responsible for translocating nascent peptides into the ER or anchoring transmembrane segments of newly synthesized proteins, were found to be enriched on sheets [ 93]. Therefore, these results suggest that the role of Climp63 in formation of sheets is likely to involve additional factors and acts as a part of an elaborate regulatory network that balances the production of sheets and tubules.

ER microtubule interactions

It is clear that proteins involved in the promotion, maintenance or stabilization of peripheral ER structures function through interactions with additional proteins or structures, and these interactions are key to proper formation of the ER network. Interestingly, several of the proteins discussed above have been shown to interact with microtubules, including Climp63 [ 91], p180 [ 94], kinectin [ 95] and STIM1 (discussed below). One important interaction discussed below is with microtubules. The ER network exhibits several dynamic interactions with microtubules that are important for determining the distribution of the ER within the cell. The two main types of interactions between the ER and microtubules are Tip Attachment Complexes (TACs) and sliding along preformed microtubules by the action of kinesin and dynein motors [ 96- 100]. In cultured cells treated with nocodazole to depolymerize microtubules, the ER retracts from the periphery [ 101], though the retraction does not occur immediately. Further investigation revealed that sliding events occurred mainly on a small subset of microtubules, modified by acetylation, that are more resistant to nocodazole treatment [ 76]. Furthermore, ER tubules can form in the absence of microtubules [ 57, 65, 68], raising many questions and leading several groups to study the interaction between ER and microtubules more in-depth.

In the past 10 years we have learned a great deal about what proteins are responsible for the intrinsic shape of the ER and how these proteins are connected to specific ER subdomains. However, we know very little about how cellular signals communicate with ER shaping proteins to change the shape of the ER in response to cellular signals.

Changes in ER structure during mitosis

During mitosis many cellular structures are dramatically remodeled to facilitate chromosome segregation. One of the most dramatic examples is changes to the microtubule cytoskeleton that occur as a result of increased microtubule dynamics caused by the action of cyclin-dependent kinases. The increase in microtubule dynamics during mitosis is important for the bipolar attachment of chromosomes to the mitotic spindle and accurate segregation to daughter cells during anaphase [ 102]. In addition to changes to the microtubule cytoskeleton, essentially all organelles change shape and function during mitosis to facilitate accurate organelle inheritance and orderly chromosome segregation. The ER undergoes dramatic shape changes during mitosis and recent studies are beginning to uncover the mechanisms linked to these structural changes.

In organisms with an open mitosis the nuclear envelope breaks down at the onset of mitosis to allow free exchange between the nucleus and cytoplasm. Nuclear envelope breakdown (NEBD) is a carefully orchestrated process that begins during mitotic prophase [ 103]. During prophase components of the nuclear pore dissociate from the pore, the nuclear lamina depolymerizes, and the membrane-bound proteins of the nuclear envelope retract into the general ER. These events free the chromosomes of nuclear lamina and membranes to facilitate chromosome condensation and segregation. In general, the events of nuclear envelope breakdown are thought to be driven by the phosphorylation of components of the NE during mitosis by various mitotic kinases, especially cyclinB:cdk1, although many molecular details are still unclear.

Concomitant with changes that occur to the nuclear envelope during NEBD the ER also begins to undergo dramatic shape changes. Changes in ER shape during mitosis have been studied in many different organisms by both light and electron microscope and these studies have resulted in a conflicting series of reports about the shape of the ER during mitosis. However, during the last few years a consensus has begun to emerge that the mitotic ER is primarily composed of sheets. Early studies using live cell microscopy in both Drosophila and C. e legans embryos demonstrated that the ER changed from a mixture of sheets and tubules to almost exclusively sheets during mitosis [ 104, 105]. Additionally, work using thin section transmission EM in HeLa cells also concluded that the majority of the ER was present in sheets throughout mitosis [ 106]. However, two studies in a variety of mammalian tissue culture cells [ 80, 107] have used both live cell microscopy and electron microscopy to suggest that the ER is primarily tubular during mitosis, and two additional studies [ 60, 108] also suggested that the ER remained tubular during mitosis and further suggested that end-on binding of ER tubules to chromatin during mitosis initiates nuclear envelope reassembly at the end of mitosis. One potential difficulty in interpreting the shape of the mitotic ER is that most cells round up during mitosis which can make acquisition of light and electron microscopy images difficult and require laborious reconstruction of the images into a three dimensional model. In addition, the mitotic ER is highly dynamic, which can complicate acquisition of live cell images during mitosis. To address these questions a series of recent studies have used both high-resolution, high-speed live cell microscopy and high-resolution EM to demonstrate that the ER is almost exclusively composed of sheets during mitosis [ 109, 110]. In addition, these studies demonstrate that the nuclear envelope reforms through the docking of ER sheets onto regions of chromatin that are isolated from spindle microtubules [ 109]. Finally, to circumvent many or the problems associated with imaging large, three dimensional cells during mitosis a recent study has examined the structure of the ER in vitro using ER reconstituted from Xenopus egg extracts [ 65]. This study convincingly demonstrated that ER formed in mitotic extracts is primarily composed of sheets while interphase ER is primarily composed of tubules. In addition, the authors demonstrated that active cyclinB:cdk1 was sufficient to convert a tubular ER into a primarily sheet based ER. Taken together all of these studies present conflicting views of the shape of the ER during mitosis, but a consensus is emerging from a wide variety of organisms that the mitotic ER is primarily composed of sheets and that the shape changes in the ER are related to changes in cyclin:cdk activity.

In addition to changes in the gross morphology of the ER during mitosis there are also dramatic changes in the distribution of proteins throughout the ER. During interphase the ER is organized into distinct domains with certain proteins defining different domains. For example, the tubule-shaping reticulon protein Rtn4 is exclusively present in the peripheral ER and excluded from the nuclear envelope [ 57, 60, 110]. In contrast, some proteins, such as the Lamin B receptor and components of the nuclear pore, are exclusively present in the nuclear envelope and are excluded from the peripheral ER [ 60, 110], while some proteins, like Sec61β, are present in all ER subdomains. However, during mitosis the NE retracts into the ER and there is nearly complete mixing of the specialized ER-shaping proteins [ 60, 110]. At the end of mitosis proteins that define the NE and peripheral ER are rapidly resorted such that they reestablish their characteristic interphase organization [ 60, 110]. In addition, it has been shown that overexpression of Rtn4 or knockdown of three reticulons (Rtn1, Rtn3, Rtn4) can either slow or speed the rate of NE reassembly at the end of mitosis, although the mechanism through which these proteins affect NE formation is currently unknown. These studies highlight the massive reorganization that takes place in the ER during mitosis and suggests that different expression levels of specific ER shaping proteins can control ER reorganization during mitosis. However, we know very little about how various ER shaping proteins are resorted to specific domains at the end of mitosis.

Two very recent studies [ 111, 112] have begun to provide insight into the specialized processes that regulate nuclear envelope reformation at the end of mitosis. Both of these studies identified a transient localization of the ESCRT-III complex to the surface of chromatin during late anaphase when the nuclear envelope is beginning to reform. ESCRT-III is best known for its role in the formation of multivesicular bodies during endocytosis, but also has well-documented roles in cytokinesis and viral budding from the plasma membrane [ 113]. Both studies demonstrated that the membrane binding and deformation properties of ESCRT-III are required for nuclear envelope formation. Additionally, interactions with the microtubule severing enzyme spastin and the ubiquitin recognition factor UFD1 are important for nuclear envelope reformation. These results demonstrate that an endosomal complex is important for regulating NE reformation and suggest that ESCRT-III could potentially play a role in additional aspects of ER dynamics.

The redistribution of ER shaping proteins during mitosis suggests that the fundamental activities of some of these proteins are modified during mitosis. For example, the mitotic ER is composed of primarily sheets, yet Rtn4, which promotes tubule formation [ 57], is distributed throughout the ER [ 60, 110]. This result suggests that the tubule-promoting activity of Rtn4 may be modified during mitosis to facilitate the tubule-to-sheet transition observed during mitosis. Inspection of large-scale phospho-proteomics studies reveals that a large number of ER-shaping proteins have identified mitosis-specific phosphorylation sites [ 114- 121]. Although none of the phosphorylation sites identified in these large-scale screens has been studied in detail their presence and specificity to mitosis suggests that these are likely to be involved in reshaping the ER during mitosis.

In support of the hypothesis that mitosis-specific phosphorylation of ER-shaping proteins regulates ER remodeling during mitosis two studies have examined this phenomenon in detail. A study of the ER sheet promoting protein Climp63 [ 51] has demonstrated mitosis-specific phosphorylation on three N-terminal residues [ 121]. Phosphorylation of Climp63 blocks the interaction of Climp63 with microtubules. Additionally, phosphomimetic mutants blocked the interaction of the ER with microtubules during interphase and resulted in an ER composed primarily of sheets, while nonphosphorylatable mutants tethered the ER to microtubules and resulted in an extremely distorted ER. These results suggest that mitotic phosphorylation of Climp63 likely blocks the interaction of the ER with microtubules and could be an important step in the tubule-to-sheet transition that occurs during mitosis. A second study examined the interaction of the ER with growing microtubule plus ends during mitosis. During interphase the ER-associated protein STIM1 interacts with the microtubule plus end-binding protein EB1 to couple ER reshaping to microtubule polymerization [ 122]. However, during mitosis the ER is excluded from the mitotic spindle and does not exhibit plus tip growth events. A recent study [ 123] has demonstrated that STIM1 is specifically phosphorylated during mitosis to control the interaction of the ER with microtubules. Specifically, phosphorylation of STIM1 blocks the interaction with the plus-end tracking protein EB1. Nonphosphorylatable mutants of STIM1, created by mutation of 10 S/T residues that block all mitotic phosphorylation, result in a recruitment of the ER throughout the spindle by restoration of the interaction of STIM1 with EB1, demonstrating that phosphorylation is a major mechanism that regulates the association of the ER with microtubules during mitosis. Interestingly, phosphorylation of STIM1 also blocks activation of SOCE, although this occurs independently of the STIM1:EB1 interaction [ 118]. Clearly much more work remains before we have a clear understanding of how cell cycle signaling cascades contribute to reshaping of the mitotic ER.

While the above studies demonstrated that phosphorylation of key proteins that link the ER to the microtubule cytoskeleton is important for excluding the ER from the spindle during mitosis a recent study demonstrated the importance of an interaction of the ER with microtubules for clearing the ER from mitotic chromatin. During mitosis the nuclear envelope is absorbed into the ER and is cleared from the surface of the chromatin, however little is known about the mechanisms that regulate ER removal from the chromatin. A recent study used a biochemical approach to identify proteins that bind to both membranes and microtubules to identify new ER proteins REEP3/4 [ 124]. The authors demonstrate that RNAi against REEP3/4 results in a failure to remove membranes from chromosomes during mitosis, resulting in chromosome segregation defects and internuclear membrane inclusions. Interestingly, the authors further demonstrate that removal of membranes from mitotic chromatin requires the interaction of REEP3/4 with microtubules. However, it is not known if REEP3/4 is subject to phosphoregulation during mitosis or if the microtubule-binding activity or REEP3/4 is required for shaping the ER during interphase. Taken together these three studies demonstrate that interaction of the ER with microtubules is a major mechanism that contributes to shape rearrangement during mitosis and that ER:microtubule interactions are regulated by mitotic phosphorylation. In addition, these studies demonstrate that the ER interacts with microtubules using many different adaptor proteins and that these different adaptor proteins serve different functions during mitosis.

Changes in ER during oocyte maturation and fertilization

One of the greatest changes during development occurs at fertilization. As in mitosis, the transition from oocyte to embryo requires many coordinated cellular changes including release from meiotic arrest, resumption of mitosis, fusion of pronuclei, activation of signaling cascades and changes in protein expression [ 125- 128]. In order for development to proceed normally, the egg must undergo the proper calcium response in order to initiate the developmental program and embryogenesis [ 129].

While the exact mechanism and conformational changes vary slightly among all organisms studied, the ER architecture in oocytes of all animals changes including Xenopus [ 130, 131], sea urchin [ 132], starfish [ 133] and mouse [ 134]. Initial studies in starfish oocytes revealed that the ER is comprised of interconnected sheets of membranes, though following germinal vesicle breakdown (GVBD), the ER sheets wrap around yolk platelets resembling a shell [ 133]. In immature mouse oocytes, large clusters were found deep within the cytoplasm [ 134]. Following GVBD, the spindle and surrounding ER migrate to the cortex leading to another round of ER reorganization into vegetally localized clusters in the metaphase II egg in addition to a finer reticular network throughout the egg [ 134, 135]. Interestingly, these steps are dependent on the microtubule network as nocodazole and inhibition of cytoplasmic dynein both prevent the ER reorganization [ 135]. Formation of the ER clusters is prevented by the depolymerization of microfilaments, but not microtubules [ 135]. Given the timing of each of these reorganizations, it seems likely that they are related to increases in cyclinB:cdk1 activity that occurs upon oocyte maturation [ 136]. These observations show an additional time in development where the ER and microtubule network interact to regulate ER structure.

In Xenopus immature oocytes, the network in both the animal (pigmented) half and vegetal (unpigemented) half appears to be uniform and consists of tubules and individual, unstacked sheets [ 130]. Additionally, the vegetal half contains annulate lamellae, stacks of sheets with membranes containing densely packed nuclear pores [ 130]. In mature eggs, the ER in the animal half is unchanged, however the annulate lamellae in the vegetal half disappeared. Interestingly, it has been proposed that the annulate lamellae share many properties with the nuclear envelope [ 137]. In place of the annulate lamellae dense, irregularly shaped ER clusters were present. The appearance of these clusters coincided with germinal vesicle breakdown. These clusters disappeared and reappeared throughout maturation and upon fertilization dispersed and permanently disappeared. The reorganization of the ER is coupled to the cell cycle as the clusters present in mature eggs contain IP ₃ receptors [ 130] and release calcium from IP ₃ channels at fertilization [ 138, 139].

Along with these changes comes a transient intracellular calcium wave, initiated during sperm entry, released from the ER and extracellular stores [ 40, 42, 140- 142]. There is one major difference in eggs of mice versus eggs of frogs. Frogs, as well as sea urchin [ 143] and starfish [ 133, 144] have a single calcium transient at fertilization [ 145]. Other animals, including mice and humans, have multiple smaller calcium transients following fertilization, and these differences may be reflected in the ER organization in mature eggs [ 145]. Mice [ 134] and frogs display ER clusters that are similar in size and location (the side opposite the meiotic spindle) and possess IP ₃ receptors [ 130, 146]. However, fertilization in mice occurs on the side with the ER clusters whereas fertilization in frogs occurs in the animal pole where the meiotic spindle is located. Therefore, the clusters may be involved in secondary calcium wave propagation. The organization of the ER network, and the reorganization throughout oogenesis, serves as a functional consequence of calcium signaling and propagation in these organisms [ 129]. We currently do not know much about the molecular mechanisms that lead to changes in ER shape during meiotic maturation and fertilization, and this should be a major are of research interest.

ER changes in response to ER stress

As seen so far, the ER is an organelle of many different functions that must be tightly regulated to carry out the proper functions. One of the most prominent functions of the ER is protein synthesis. Even with several chaperones and folding enzymes in place, an accumulation of unfolded or misfolded proteins in the lumen of the ER can occur. When the cell undergoes this type of stress there are several things that must occur to retain balance and proper function, including translational inhibition, degradation of unfolded or misfolded proteins, and an increase in the production of chaperones and folding enzymes to restore normal function of the ER and the cell. If the balance is not restored it can lead to cell death or apoptosis [ 147], therefore achieving normal function is critical to the survival of the cell.

As discussed above, once a peptide destined for secretion has entered the lumen of the cell, there are several modifications that occur, including N-linked glycosylation, disulfide bond formation and oligomerization [ 3]. N-linked glycosylation can occur co-translationally as the protein is translocated into the ER lumen. The oligosaccharyltransferase (OST) can modify the Asparagine within the Asn-X-Ser/Thr sequence once it has traversed approximately 13 amino acids into the ER lumen [ 148], which improves the kinetics and thermodynamics of folding for proteins [ 149, 150]. Misfolding can occur due to the unique environment of the lumen and the high protein concentration of both newly synthesized proteins, proteins ready for secretion and proteins that act as molecular chaperones and folding enzymes. Logistically, due to the high protein concentration and packing in the lumen, the folding enzymes must first identify and find the proper target protein for folding to take place. If proteins are not modified correctly, the lack of glucose residues is recognized by the ER and proteins including UDP-glucose:glycoprotein glucosyltransferase (UGGT) in an attempt to re-glycosylate the protein [ 151- 153]. If the normal folding process is not restored, hydrophobic residues are exposed and bound by Grp78, accumulation of these proteins occurs and the unfolded protein response (UPR) is activated [ 154, 155]. The first action of the UPR is to increase ER abundance to accommodate the needs of the cell to properly fold the proteins, leading to an expansion of the ER through the generation of sheets [ 156] and an increase in the ER folding machinery.

The UPR consists of three parallel branches that are activated upon stress and include inositol requiring enzyme 1 (IRE1) by nonconventional splicing, double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) through translational control by phosphorylating eIF2α, and activating transcription factor 6 (ATF6) through regulated proteolysis [ 155]. Briefly, activation of these pathways lead to production of b-ZIP transcription factors that activate UPR genes [ 155]. First, ER-resident IRE1, a transmembrane endoribonuclease, mediates the post-transcriptional, non-canonical splicing of XBP1 mRNA that is localized to the ER [ 157- 159] and encodes a transcription factor involved in upregulating additional stress response genes. Additionally, the nuclease activity of IRE1 is involved in degradation of a subset of ER-associated RNAs in a process known as IRE1-dependent decay (RIDD) [ 160, 161]. The cell has evolved this mechanism to reduce the translational load on the ER by removing mRNAs that otherwise would be translated, and may be one way for the cell to upregulate stress-response genes that are needed in the UPR. Although it is clear that ER-stress leads to large scale changes in the protein and RNA content of the ER, it is not yet clear if this leads to immediate structural reorganization in order to accommodate the new needs of the organelle. In addition, it is not yet clear if activation of stress-responsive signaling pathways leads to the modification of intrinsic structural components of the ER. Interestingly, it has been observed that splicing of XBP1 is activated during meiosis in both Xenopus and budding yeast [ 162, 163], suggesting that changes in ER structure during meiosis could be linked to the ER stress response. These would both be interesting avenues of future research exploring structural changes in the ER in response to cellular signaling cues.

Closing remarks

The ER is a complex organelle that plays a pivotal role in protein and lipid synthesis, calcium storage and stress response. Changes in structure in response to cell cycle or developmental state render this organelle highly dynamic. Several proteins play a role in the proper formation of the different structures of the peripheral ER including the nuclear envelope, sheets and tubules. Regulation exists at multiple steps in the formation and maintenance of these structures, and the ratios of these structures are very different in cells of different functions. In general, cells involved in synthesizing large amounts of protein have higher ratios of sheets, whereas cells involved in lipid synthesis or signaling with other organelles would have higher ratios of tubules. The generation of these structures relies on a myriad of proteins, involved in either structural aspects of ER morphology by directly affecting the phospholipid bilayer and curvature of membranes or mediating interactions with other organelles or the cytoskeleton. In addition, proteins with other functions, including nucleases and GTPases, also play a role in network formation. Recent work has begun to connect our knowledge of the proteins that provide the fundamental shape of the ER to signaling pathways, but much work remains to be done to understand how developmental, cell cycle, and stress pathways change the fundamental shape of the ER in different circumstances. Recent work on several different human diseases has highlighted a role for several different ER-shaping proteins in diverse diseases such as Alzheimer's and Hereditary Spastic Paraplegia (HSP) [reviewed in 7]. The strong link of ER-shaping proteins to hereditary human diseases highlights the need for further research into the basic biology of the ER and how this biology changes in response to changes in cellular environment.

References

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Articles from Cellular and Molecular Life Sciences are provided here courtesy of Springer

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The endoplasmic reticulum: structure, function and response to cellular signaling

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The endoplasmic reticulum (ER) is a large, dynamic structure that serves many roles in the cell including calcium storage, protein synthesis and lipid metabolism. The diverse functions of the ER are performed by distinct domains; consisting of tubules, ...

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The nuclear membrane acts as a barrier that prevents a passage for molecules between the nucleus and cytoplasm.

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protects the nucleus’s content; the DNA of the cell

Carries proteins which are crucial in the organization of DNA and to control genes

Only eukaryotes have nuclear membranes since prokaryotes do not have a nucleus

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"This structure alone creates what is known as a selectively permeable barrier, one that only allows substances meeting certain physical criteria to pass through it. In the case of the cell membrane, only relatively small, nonpolar materials can move through the lipid bilayer at biologically relevant rates (remember, the lipid tails of the membrane are nonpolar)."

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Page ID

8531

1. Transport across the membrane

1. Relative permeability

2. Membrane permeability coefficients

3. Energetics of transport

1. Passive transport

1. Diffusion

2. Factors that affect diffusion

2. Facilitated transport

1. Channels

2. Carrier proteins

4. Active transport

1. Moving against a gradient

2. Carrier proteins for active transport

3. Primary active transport

4. Secondary active transport (co-transport)

2. Osmosis

1. Mechanism

2. Tonicity

1. Hypotonic solutions

2. Hypertonic solutions

3. Isotonic solutions

3. Tonicity in living systems

Transport across the membrane

Design challenge problem and subproblems
General Problem: The cell membrane must simultaneously act as a barrier between "IN" and "OUT" and control specifically which substances enter and leave the cell and how quickly and efficiently they do so. Subproblems: The chemical properties of molecules that must enter and leave the cell are highly variable. Some subproblems associated with this are: (a) Large and small molecules or collections of molecules must be able to pass across the membrane. (b) Both hydrophobic and hydrophilic substances must have access to transport. (c) Substances must be able to cross the membrane with and against concentration gradients. (d) Some molecules look very similar (e.g. Na⁺ and K⁺) but transport mechanisms must still be able to distinguish between them.
Energy story perspective

Transport across a membrane can be considered from an energy story perspective; it is a process after all. For instance, at the beginning of the process a generic substance X may be either on the inside or outside of the cell. At the end of the process, the substance will be on the opposite side from which it started.

e.g. X(in) ---> X(out),

where in and out refer to inside the cell and outside the cell, respectively.

At the beginning the matter in the system might be a very complicated collection of molecules inside and outside of the cell but with one molecule of X more inside the cell than out. At the end, there is one more molecule of X on the outside of the cell and one less on the inside. The energy in the system at the beginning is stored largely in the molecular structures and their motions and in electrical and chemical concentration imbalances across the cell membrane. The transport of X out of the cell will not change the energies of the molecular structures significantly but it will change the energy associated with the imbalance of concentration and or charge across the membrane. That is the transport will, like all other reactions, be either exergonic or endergonic. Finally, some mechanism or sets of mechanisms of transport will need to be described.

Selective permeability

One of the great wonders of the cell membrane is its ability to regulate the concentration of substances inside the cell. These substances include: ions such as Ca ²⁺, Na⁺, K⁺, and Cl ⁻; nutrients including sugars, fatty acids, and amino acids; and waste products, particularly carbon dioxide (CO ₂), which must leave the cell.

The membrane's lipid bilayer structure provides the first level of control. The phospholipids are tightly packed, and the membrane has a hydrophobic interior. This structure alone creates what is known as a selectively permeable barrier, one that only allows substances meeting certain physical criteria to pass through it. In the case of the cell membrane, only relatively small, nonpolar materials can move through the lipid bilayer at biologically relevant rates (remember, the lipid tails of the membrane are nonpolar).

Selective permeability of the cell membrane refers to its ability to differentiate between different types of molecules, only allowing some molecules through while blocking others. Some of this selective property stems from the intrinsic diffusion rates for different molecules across a membrane. A second factor affecting the relative rates of movement of various substances across a biological membrane is activity of various protein-based membrane transporters, both passive and active, that will be discussed in more detail in subsequent sections. First, we take on the notion of intrinsic rates of diffusion across the membrane.

Relative permeability

The fact that different substances might cross a biological membrane at different rates should be relatively intuitive. There are differences in the mosaic composition of membranes in biology and differences in the sizes, flexibility, and chemical properties of molecules so it stands to reason that the permeability rates vary. It is a complicated landscape. The permeability of a substance across a biological membrane can be measured experimentally and the rate of movement across a membrane can be reported in what are known as membrane permeability coefficients.

Membrane permeability coefficients

Below, a variety of compounds are plotted with respect to their membrane permeability coefficients (MPC) as measured against a simple biochemical approximation of a real biological membrane. The reported permeability coefficient for this system is the rate at which simple diffusion through a membrane occurs and is reported in units of centimeters per second (cm/s). The permeability coefficient is proportional to the partition coefficient and is inversely proportional to the membrane thickness.

It is important that you are able to read and interpret the diagram below. The larger the coefficient, the more permeable the membrane is to the solute. For example, hexanoic acid is very permeable, a MPC of 0.9; acetic acid, water, and ethanol have MPCs between 0.01 and 0.001, and they are less permeable than hexanoic acid. Where as ions, such as sodium (Na⁺), have an MPC of 10⁻¹², and cross the membrane at a comparatively slow rate.

While there are certain trends or chemical properties that can be roughly associated with different compound permeability (small thing go through "fast", big things "slowly", charged things not at all etc.), we caution against over-generalizing. The molecular determinants of membrane permeability are complicated and involve numerous factors including: the specific composition of the membrane, temperature, ionic composition, hydration; the chemical properties of the solute; the potential chemical interactions between the solute in solution and in the membrane; the dielectric properties of materials; and the energy trade-offs associated with moving substances into and out of various environments. So, in this class, rather than try to apply "rules" and try to develop too many arbitrary "cut-offs", we will strive to develop a general sense of some properties that can influence permeability and leave the assignment of absolute permeability to experimentally reported rates. In addition, we will also try to minimize the use of vocabulary that depends on a frame of reference. For instance, saying that compound A diffuses "quickly" or "slowly" across a bilayer only means something if the terms "quickly" or "slowly" are numerically defined or the biological context is understood.

Energetics of transport

All substances that move through the membrane do so by one of two general methods, which are categorized based on whether or not the transport process is exergonic or endergonic. Passive transport is the exergonic movement of substances across the membrane. In contrast, active transport is the endergonic movement of substances across the membrane that is coupled to an exergonic reaction.

Passive transport

Passive transport does not require the cell to expend energy. In passive transport, substances move from an area of higher concentration to an area of lower concentration, down their concentration gradient . Depending on the chemical nature of the substance, different processes may be associated with passive transport.

Facilitated transport

In facilitated transport, also called facilitated diffusion, materials diffuse across the plasma membrane with the help of membrane proteins. A concentration gradient exists that allows these materials to diffuse into or out of the cell without expending cellular energy. In the case that the materials are ions or polar molecules (compounds that are repelled by the hydrophobic parts of the cell membrane), facilitated transport proteins help shield these materials from the repulsive force of the membrane, allowing them to diffuse into the cell.

Note: possible discussion

Active transport

Active transport mechanisms require the use of the cell's energy, usually in the form of adenosine triphosphate (ATP). If a substance must move into the cell against its concentration gradient-that is, if the concentration of the substance inside the cell is greater than its concentration in the extracellular fluid (and vice versa)-the cell must use energy to move the substance. Some active transport mechanisms move small-molecular weight materials, such as ions, through the membrane. Other mechanisms transport much larger molecules.

Moving against a gradient

To move substances against a concentration or electrochemical gradient, the cell must use energy. This energy is harvested from ATP generated through the cell's metabolism. Active transport mechanisms, collectively called pumps, work against electrochemical gradients. Small substances constantly pass through plasma membranes. Active transport maintains concentrations of ions and other substances needed by living cells in the face of these passive movements. Much of a cell's supply of metabolic energy may be spent maintaining these processes. (Most of a red blood cell's metabolic energy is used to maintain the imbalance between exterior and interior sodium and potassium levels required by the cell.) Because active transport mechanisms depend on a cell's metabolism for energy, they are sensitive to many metabolic poisons that interfere with the supply of ATP.

Two mechanisms exist for the transport of small-molecular weight material and small molecules. Primary active transport moves ions across a membrane and creates a difference in charge across that membrane, which is directly dependent on ATP. Secondary active transport describes the movement of material that is due to the electrochemical gradient established by primary active transport that does not directly require ATP.

Carrier proteins for active transport

An important membrane adaption for active transport is the presence of specific carrier proteins or pumps to facilitate movement: there are three types of these proteins or transporters. A uniporter carries one specific ion or molecule. A symporter carries two different ions or molecules, both in the same direction. An antiporter also carries two different ions or molecules, but in different directions. All of these transporters can also transport small, uncharged organic molecules like glucose. These three types of carrier proteins are also found in facilitated diffusion, but they do not require ATP to work in that process. Some examples of pumps for active transport are Na⁺-K⁺ ATPase, which carries sodium and potassium ions, and H⁺-K⁺ ATPase, which carries hydrogen and potassium ions. Both of these are antiporter carrier proteins. Two other carrier proteins are Ca ²⁺ATPase and H⁺ ATPase, which carry only calcium and only hydrogen ions, respectively. Both are pumps.

Primary active transport

In primary active transport, the energy is often - though not exclusively - derived directly from the hydrolysis of ATP. Often, primary active transport, such as that shown below, which functions to transport sodium and potassium ions allows secondary active transport to occur (discussed in the section below). The second transport method is still considered active because it depends on the use of energy from the primary transport.

One of the most important pumps in animal cells is the sodium-potassium pump (Na⁺-K⁺ ATPase), which maintains the electrochemical gradient (and the correct concentrations of Na⁺and K⁺) in living cells. The sodium-potassium pump moves K⁺ into the cell while moving Na⁺ out at the same time, at a ratio of three Na⁺ for every two K⁺ ions moved in. The Na⁺-K⁺ ATPase exists in two forms depending on its orientation to the interior or exterior of the cell and its affinity for either sodium or potassium ions. The process consists of the following six steps.

1. With the enzyme oriented towards the interior of the cell, the carrier has a high affinity for sodium ions. Three ions bind to the protein.

2. ATP is hydrolyzed by the protein carrier and a low-energy phosphate group attaches to it.

3. As a result, the carrier changes shape and re-orients itself towards the exterior of the membrane. The protein's affinity for sodium decreases and the three sodium ions leave the carrier.

4. The shape change increases the carrier's affinity for potassium ions, and two such ions attach to the protein. Subsequently, the low-energy phosphate group detaches from the carrier.

5. With the phosphate group removed and potassium ions attached, the carrier protein repositions itself towards the interior of the cell.

6. The carrier protein, in its new configuration, has a decreased affinity for potassium, and the two ions are released into the cytoplasm. The protein now has a higher affinity for sodium ions, and the process starts again.

Several things have happened as a result of this process. At this point, there are more sodium ions outside of the cell than inside and more potassium ions inside than out. For every three ions of sodium that move out, two ions of potassium move in. This results in the interior being slightly more negative relative to the exterior. This difference in charge is important in creating the conditions necessary for the secondary process. The sodium-potassium pump is, therefore, an electrogenic pump (a pump that creates a charge imbalance), creating an electrical imbalance across the membrane and contributing to the membrane potential.

Secondary active transport (co-transport)

Secondary active transport brings sodium ions, and possibly other compounds, into the cell. As sodium ion concentrations build outside of the plasma membrane because of the action of the primary active transport process, an electrochemical gradient is created. If a channel protein exists and is open, the sodium ions will be pulled through the membrane. This movement is used to transport other substances that can attach themselves to the transport protein through the membrane. Many amino acids, as well as glucose, enter a cell this way. This secondary process is also used to store high energy hydrogen ions in the mitochondria of plant and animal cells for the production of ATP. The potential energy that accumulates in the stored hydrogen ions is translated into kinetic energy as the ions surge through the channel protein ATP synthase, and that energy is used to convert ADP into ATP.

Osmosis

Osmosis is the movement of water through a semipermeable membrane according to the concentration gradient of water across the membrane, which is inversely proportional to the concentration of solutes. While diffusion transports material across membranes and within cells, osmosis transports only water across a membrane and the membrane limits the diffusion of solutes in the water. Not surprisingly, the aquaporins that facilitate water movement play a large role in osmosis, most prominently in red blood cells and the membranes of kidney tubules.

Mechanism

Osmosis is a special case of diffusion. Water, like other substances, moves from an area of high concentration to one of low concentration. An obvious question is what makes water move at all? Imagine a beaker with a semipermeable membrane separating the two sides or halves. On both sides of the membrane the water level is the same, but there are different concentrations of a dissolved substance, or solute, that cannot cross the membrane (otherwise the concentrations on each side would be balanced by the solute crossing the membrane). If the volume of the solution on both sides of the membrane is the same, but the concentrations of solute are different, then there are different amounts of water, the solvent, on either side of the membrane.

To illustrate this, imagine two full glasses of water. One has a single teaspoon of sugar in it, whereas the second one contains one-quarter cup of sugar. If the total volume of the solutions in both cups is the same, which cup contains more water? Because the large amount of sugar in the second cup takes up much more space than the teaspoon of sugar in the first cup, the first cup has more water in it.

Returning to the beaker example, recall that it has a mixture of solutes on either side of the membrane. A principle of diffusion is that the molecules move around and will spread evenly throughout the medium if they can. However, only the material capable of getting through the membrane will diffuse through it. In this example, the solute cannot diffuse through the membrane, but the water can. Water has a concentration gradient in this system. Thus, water will diffuse down its concentration gradient, crossing the membrane to the side where it is less concentrated. This diffusion of water through the membrane-osmosis-will continue until the concentration gradient of water goes to zero or until the hydrostatic pressure of the water balances the osmotic pressure. Osmosis proceeds constantly in living systems.

Tonicity

Tonicity describes how an extracellular solution can change the volume of a cell by affecting osmosis. A solution's tonicity often directly correlates with the osmolarity of the solution. Osmolarity describes the total solute concentration of the solution. A solution with low osmolarity has a greater number of water molecules relative to the number of solute particles; a solution with high osmolarity has fewer water molecules with respect to solute particles. In a situation in which solutions of two different osmolarities are separated by a membrane permeable to water, though not to the solute, water will move from the side of the membrane with lower osmolarity (and more water) to the side with higher osmolarity (and less water). This effect makes sense if you remember that the solute cannot move across the membrane, and thus the only component in the system that can move-the water-moves along its own concentration gradient. An important distinction that concerns living systems is that osmolarity measures the number of particles (which may be molecules) in a solution. Therefore, a solution that is cloudy with cells may have a lower osmolarity than a solution that is clear if the second solution contains more dissolved molecules than there are cells.

Hypotonic solutions

Three terms-hypotonic, isotonic, and hypertonic-are used to relate the osmolarity of a cell to the osmolarity of the extracellular fluid that contains the cells. In a hypotonicsituation, the extracellular fluid has lower osmolarity than the fluid inside the cell, and water enters the cell (in living systems, the point of reference is always the cytoplasm, so the prefix hypo- means that the extracellular fluid has a lower concentration of solutes, or a lower osmolarity, than the cell cytoplasm). It also means that the extracellular fluid has a higher concentration of water in the solution than does the cell. In this situation, water will follow its concentration gradient and enter the cell.

Hypertonic solutions

As for a hypertonic solution, the prefix hyper- refers to the extracellular fluid having a higher osmolarity than the cell's cytoplasm; therefore, the fluid contains less water than the cell does. Because the cell has a relatively higher concentration of water, water will leave the cell.

Isotonic solutions

In an isotonic solution, the extracellular fluid has the same osmolarity as the cell. If the osmolarity of the cell matches that of the extracellular fluid, there will be no net movement of water into or out of the cell, although water will still move in and out. Blood cells and plant cells in hypertonic, isotonic, and hypotonic solutions take on characteristic appearances.

Connection

Tonicity in living systems

In a hypotonic environment, water enters a cell, and the cell swells. In an isotonic condition, the relative concentrations of solute and solvent are equal on both sides of the membrane. There is no net water movement; therefore, there is no change in the size of the cell. In a hypertonic solution, water leaves a cell and the cell shrinks. If either the hypo- or hyper- condition goes to excess, the cell's functions become compromised, and the cell may be destroyed.

A red blood cell will burst, or lyse, when it swells beyond the plasma membrane's capability to expand. Remember, the membrane resembles a mosaic, with discrete spaces between the molecules composing it. If the cell swells, and the spaces between the lipids and proteins become too large, and the cell will break apart.

In contrast, when excessive amounts of water leave a red blood cell, the cell shrinks, or crenates. This has the effect of concentrating the solutes left in the cell, making the cytosol denser and interfering with diffusion within the cell. The cell's ability to function will be compromised and may also result in the death of the cell.

Various living things have ways of controlling the effects of osmosis-a mechanism called osmoregulation. Some organisms, such as plants, fungi, bacteria, and some protists, have cell walls that surround the plasma membrane and prevent cell lysis in a hypotonic solution. The plasma membrane can only expand to the limit of the cell wall, so the cell will not lyse. In fact, the cytoplasm in plants is always slightly hypertonic to the cellular environment, and water will always enter a cell if water is available. This inflow of water produces turgor pressure, which stiffens the cell walls of the plant. In nonwoody plants, turgor pressure supports the plant. Conversely, if the plant is not watered, the extracellular fluid will become hypertonic, causing water to leave the cell. In this condition, the cell does not shrink because the cell wall is not flexible. However, the cell membrane detaches from the wall and constricts the cytoplasm. This is called plasmolysis. Plants lose turgor pressure in this condition and wilt.

Figure 11. Without adequate water, the plant on the left has lost turgor pressure, visible in its wilting; the turgor pressure is restored by watering it (right). (credit: Victor M. Vicente Selvas)

Tonicity is a concern for all living things. For example, paramecia and amoebas, which are protists that lack cell walls, have contractile vacuoles. This vesicle collects excess water from the cell and pumps it out, keeping the cell from bursting as it takes on water from its environment.
Figure 12. A paramecium's contractile vacuole, here visualized using bright field light microscopy at 480x magnification, continuously pumps water out of the organism's body to keep it from bursting in a hypotonic medium. (credit: modification of work by NIH; scale-bar data from Matt Russell)
Many marine invertebrates have internal salt levels matched to their environments, making them isotonic with the water in which they live. Fish, however, must spend approximately five percent of their metabolic energy maintaining osmotic homeostasis. Freshwater fish live in an environment that is hypotonic to their cells. These fish actively take in salt through their gills and excrete diluted urine to rid themselves of excess water. Saltwater fish live in the reverse environment, which is hypertonic to their cells, and they secrete salt through their gills and excrete highly concentrated urine.

In vertebrates, the kidneys regulate the amount of water in the body. Osmoreceptors are specialized cells in the brain that monitor the concentration of solutes in the blood. If the levels of solutes increase beyond a certain range, a hormone is released that retards water loss through the kidney and dilutes the blood to safer levels. Animals also have high concentrations of albumin, which is produced by the liver, in their blood. This protein is too large to pass easily through plasma membranes and is a major factor in controlling the osmotic pressures applied to tissues.

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15.3: Membrane Transport with Selective Permeability

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Page ID 8531 Transport across the membrane Relative permeability Membrane permeability coefficients Energetics of transport Passive transport Diffusion Factors that affect diffusion Facilitated transport Channels Carrier proteins Active transport Moving against a gradient Carrier proteins for active transport Primary active transport Secondary active transport (co-transport) Osmosis Mechanism Tonicity Hypotonic solutions Hypertonic solutions Isotonic solutions Tonicity in living systems Design challenge problem and subproblems General Problem: The cell membrane must simultaneously act as a barrier between "IN" and "OUT" and control specifically which substances enter and leave the cell and how quickly and efficiently they do so.

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Mitochondria are membrane-bound cell organelles (mitochondrion, singular) that generate most of the chemical energy needed to power the cell's biochemical reactions. Chemical energy produced by the mitochondria is stored in a small molecule called adenosine triphosphate (ATP).

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Mitochondria are membrane-bound organelles, but they're membrane-bound with two different membranes. And that's quite unusual for an intercellular organelle. Those membranes function in the purpose of mitochondria, which is essentially to produce energy. That energy is produced by having chemicals within the cell go through pathways, in other words, be converted. And the process of that conversion produces energy in the form of ATP, because the phosphate is a high-energy bond and provides energy for other reactions within the cell. So the mitochondria's purpose is to produce that energy. Some different cells have different amounts of mitochondria because they need more energy. So for example, the muscle has a lot of mitochondria, the liver does too, the kidney as well, and to a certain extent, the brain, which lives off of the energy those mitochondria produce. So if you have a defect in the pathways that the mitochondria usually functions with, you're going to have symptoms in the muscle, in the brain, sometimes in the kidneys as well; many different types of symptoms. And we probably don't know all of the different diseases that mitochondrial dysfunction causes.

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Mitochondria are membrane-bound cell organelles (mitochondrion, singular) that generate most of the chemical energy needed to power the cell's biochemical reactions.

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Flagella move via a spining motion like a drive shaft. Flagella exist as a way to help difrent types of cells move around like the sperm cell seen above.

https://www.youtube.com/watch?v=PBrxh3N8CuA

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The lysosomes are a membrane organelle that contain enzymes that break down materials