Within membranes, the ability of microdomains to sequester specific proteins and exclude others makes them ideally suited to spatially organize cellular pathways. For instance, numerous studies of the distribution of SNARE proteins in various cell types suggest that SNAREs partially associate with detergent resistant, cholesterol-enriched microdomains Palmitoylation appears to be the major targeting signal in these microdomains, as in the case of SNAP, although it is likely that other elements contribute to the enrichment of constituents of the exocytotic machinery within these cholesterol microdomains.
However despite intense research there is still little known about what lipid or protein molecules are actually present at sites of exocytosis. Up to 20 proteins potentially involved in regulated exocytosis have been reported to bind PtdIns 4,5 P 2 Using immunogold labeling of plasma membrane sheets combined with spatial point pattern analysis, we recently observed that PtdIns 4,5 P 2 microdomains co-localize with SNARE clusters and docked secretory granules Translocation of the PtdIns 4,5 P 2 -binding protein annexin A2 to the plasma membrane following cell stimulation is a hallmark of chromaffin cell exocytosis Annexin A2 plays an essential role in calcium-regulated exocytosis by promoting PtdIns 4,5 P 2 and cholesterol-enriched domains containing SNAREs in the vicinity of docked granules 9 , Altogether these observation raise the notion that functional exocytotic sites defined by specific lipids such as cholesterol, GM1, and PtdIns 4,5 P 2 are able to recruit and sequester components to build a machine that drives fast and efficient membrane fusion Figure 1.
Figure 1. Model highlighting the importance of lipids for membrane fusion. Molecular details of how PtdIns 4,5 P 2 forms a platform for vesicle recruitment have recently been proposed Like PtdIns 4,5 P 2 , these anionic lipids can probably recruit syntaxin-1A, and it is tempting to propose that the recruitment of syntaxin isoforms may depend on the type of lipid present.
Furthermore, the fact that these lipids can be quickly converted into different forms using kinases, lipases, and phosphatases, such a protein-recruiting mechanism offers a supplementary level of control to adapt the exocytotic machinery to the physiological demands put on the cell.
Finally, using super-resolution optical techniques and fluorescence lifetime imaging microscopy, it was shown that distinct t-SNARE intermediate states on the plasma membrane can be patterned by the underlying lipid environment Undoubtedly these high-resolution imaging techniques will be useful to determine how lipids contribute to the organization of the exocytotic platform. PtdIns 4,5 P 2 directly binds to a large subset of proteins from the exocytotic machinery.
Finally, PtdIns 4,5 P 2 controls actin polymerization by modulating the activity and targeting of actin regulatory proteins. Indeed the activity of the actin-binding proteins scinderin and gelsolin, two F-actin severing proteins that are constituents of the exocytotic machinery, is regulated by PtdIns 4,5 P 2 A transient increase in PIP2 levels is sufficient to promote the mobilization and recruitment of secretory vesicles to the plasma membrane PIP2 therefore links exocytosis and the actin cytoskeleton by coordinating the actin-based delivery of secretory vesicles to the exocytotic sites.
Diacylglycerol production through hydrolysis of PtdIns 4,5 P 2 by phospholipase C is mandatory for exocytosis DAG is essential in the priming of exocytosis, owing to the activation of protein kinase C and Munc13, which then modulate the function of syntaxin-1A This pathway is essential for exocytosis as inhibition of DAG lipase blocks exocytosis PA directly activates these proteins, but evidence that this activation directly contributes to exocytosis remains scarce.
PA is also an essential cofactor of phosphatidylinositolphosphate 5-kinase, which produces PtdIns 4,5 P 2 , suggesting a possible positive feedback loop in the synthesis of PA and PtdIns 4,5 P 2 Although no direct evidence in neuroendocrine systems have shown that PA directly regulates the assembly or the function of the minimal fusion machinery, in vitro reconstituted fusion assays with purified yeast vacuolar SNAREs do so.
Interestingly, omega-3 and omega-6 fatty acids, which play important roles in human health, have be shown to recapitulate this in vitro effect of arachidonic acid on SNARE complex formation, suggesting that syntaxins may represent crucial targets of polyunsaturated lipids In other words, polyunsaturated lipids may physiologically regulate SNARE complex assembly and thus exocytosis.
Along this same line, sphingosine a releasable backbone of sphingolipids, activates vesicular synaptobrevin facilitating the assembly of SNARE complexes required for membrane fusion It is however important to note that the effects of arachidonic acid and sphingosine observed in these studies are all achieved near or at the CMC value for these lipids, treatments that may also lead to membrane disorganization like detergent action.
The most widely accepted model for membrane fusion, the stalk pore model proposes that the merging of cis contacting monolayers gives rise to a negatively curved lipid structure called a stalk. The structure of this stalk depends on the composition of the cis monolayers the outer leaflet of the vesicle and the inner leaflet of the plasma membrane.
This model implies that cone-shaped lipids such as cholesterol, DAG, or PA, which have intrinsic negative curvatures, in the cis leaflets of contacting bilayers would enhance membrane fusion Vice versa inverted cone-shaped lipids such as PS, gangliosides, or lysophospholipids should prevent membrane fusion in the cis leaflets, but promote fusion when present in the outer leaflets Interestingly, GM1 was found enriched in the outer leaflet of the plasma membrane at the sites of exocytosis in stimulated chromaffin cells 9.
These GM1 domains may induce positive membrane curvature in the outer leaflet 55 , thereby promoting fusion Figure 1. Reconstituted fusion assays and direct addition of lipids on cultured cells validate the concept that PA, DAG, and cholesterol might promote membrane fusion by changing the spontaneous curvature of membranes [reviewed in 8 ].
At physiological concentrations, PtdIns 4,5 P 2 inhibits SNARE-dependent liposome fusion 45 , most likely due to its intrinsic positive curvature-promoting properties. However, PtdIns 4,5 P 2 has been described to be converted from an inverted cone-shaped structure to a cone-shaped form in the presence of calcium Thus, in stimulated cells, a local accumulation of PA and PtdIns 4,5 P 2 at granule docking sites where GM1 is in the outer leaflets may well have a synergistic effect on membrane curvature and promote fusion Figure 1.
In an alternate mode of changing membrane topology, synaptotagmin has been proposed to facilitate membrane fusion by phase separating PS, a process that is expected to locally buckle bilayers and disorder lipids due to the curvature tendencies of PS It is worth to mention that most of lipid mentioned in this review, also have the ability to flip from one leaflet to another.
How this flipping is regulated and how it affects curvature remains an unsolved issue. However, it is likely that the ability of these lipids to interact with the fusion machinery largely controls these flipping properties. As illustrated in this review, a given lipid can play multiple functions, acting either individually or successively or even simultaneously in concert with other lipids. At the same time, the rapid enzymatic production and degradation of lipids at exocytotic sites allows the cell to remain flexible: by changing the lipid levels, physiological function can be modified within seconds or minutes without the need for protein synthesis or degradation.
Over the last decade, in vitro reconstituted membrane fusion combined with precise methods to quantify specific lipid species and improved molecular and pharmacological tools to manipulate cellular levels of a given lipid, have lead to a better understanding of the capacities of lipids to promote exocytosis at different steps of the process.
For different kinds of vesicles in different cell types, it is likely that the local lipid environment may differentially regulate fusion pore formation, enlargement, and duration, which may in part explain the great variety of fusion kinetics observed in vivo. Finally, lipids could also contribute to the tight coupling between exocytosis and the early stages of membrane retrieval and endocytosis as highlighted in a review of this issue Houy et al.
Undoubtedly, the next challenge will be to follow individual lipid dynamics at the speed of pore formation and expansion. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We wish to thank Dr. Nancy Grant for critical reading of the manuscript. Biochim Biophys Acta — Jahn R, Fasshauer D. Molecular machines governing exocytosis of synaptic vesicles.
Nature —7. Nature — SNAREpins: minimal machinery for membrane fusion. Cell 92 — CrossRef Full Text. Reconstitution of the vital functions of Munc18 and Munc13 in neurotransmitter release. Science —5. Martin TF. Role of PI 4,5 P 2 in vesicle exocytosis and membrane fusion.
Subcell Biochem 59 — Phospholipases and fatty acid signalling in exocytosis. J Physiol — Lipid dynamics in exocytosis. Cell Mol Neurobiol 30 — Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles. Cells of the immune system consistently destroy pathogens by essentially "eating" them. Some molecules or particles are just too large to pass through the plasma membrane or to move through a transport protein.
So cells use two other active transport processes to move these macromolecules large molecules into or out of the cell. Vesicles or other bodies in the cytoplasm move macromolecules or large particles across the plasma membrane. There are two types of vesicle transport, endocytosis and exocytosis illustrated in Figure below. Both processes are active transport processes, requiring energy. Illustration of the two types of vesicle transport, exocytosis and endocytosis.
Endocytosis is the process of capturing a substance or particle from outside the cell by engulfing it with the cell membrane. The membrane folds over the substance and it becomes completely enclosed by the membrane. At this point a membrane-bound sac, or vesicle, pinches off and moves the substance into the cytosol. There are two main kinds of endocytosis:. Transmission electron microscope image of brain tissue that shows pinocytotic vesicles.
Pinocytosis is a type of endocytosis. Provide feedback to your librarian. If you have any questions, please do not hesitate to reach out to our customer success team. Login processing Chapter 5: Membranes and Cellular Transport. Chapter 1: Scientific Inquiry. Chapter 2: Chemistry of Life. Chapter 3: Macromolecules. Chapter 4: Cell Structure and Function. Chapter 6: Cell Signaling. Chapter 7: Metabolism. Chapter 8: Cellular Respiration. Chapter 9: Photosynthesis. Chapter Cell Cycle and Division.
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