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Shape-structure concept of lipid polymorphism

Traffic 1:605

Speculative models for lipid involvement in membrane fission. Vesicles pinching off involves extremes of curvature, both concave and convex. This may involve lipids with conical or inverted conical shapes, which promote curvature.

Traffic 1: 605

Kompartmentace metabolismu lipidů v rostlinné buňce

U rostlin spolupracují dvě dráhy syntézy membránových lipidů –I. Plastidová („prokaryotická“) a II. ER („eukaryotická“ užívá mastných kyselin z plastidů)

Flipáza působí asymetrii v membránové dvouvrstvě

Stěhování membránových lipidů hydrofilním prostředím buňky je pak umožňováno aktivitou řady

lipidy přenášejících bílkovin (lipid transfer protein LTP).

cis = vazba působí deformaci

Př. trans = která se ovšem prakticky nevyskytuje

Fosfatidylcholin

V reakci na chlad rostliny optimalizují složení membrán tak, aby nehrozil nežádoucí fázový přechod (Tm) do gelu a byla zachována optimální tekutost.

1. Zkracování řetězců mastných kyselin.2. Zvyšování počtu dvojných vazeb –desaturace.3. Zvětšování velikosti a náboje polárních skupin „hlavy“

V oleosomech jako zásobní! lipid.

STEROLY fungují jako „pufry“ membránové tekutosti.

A – při nízké teplotě zvyšují tekutost tím, že brání agregaci/gelovatění fosfolipidů.B – při vysoké teplotě snižují tekutost interferencí s volným ohýbáním řetězců mastných kyselin.

Fluid Bilayer View of Membrane Structure

Lodish 4

Single-spanning Membrane Protein: 1 alpha Helix

Lodish 4

K překonání tloušťkymembrány stačí něco víc než 20AA.Tlouťka membrány během sekrece (ER,GA,exoc.) vzrůstá.

Topology of 7-spanning protein: Rhodopsin as a prototype.

Each membrane spanning segment is generally very hydrophobic and can be predicted by a peak, ~20 amino acids long, in a hydropathy plot.Proteins that span the membrane many times often have a more hydrophilic environment in the center, so that each α−helical transmembrane segment has a hydrophobic face towards the lipid bilayer and a more hydrophilic face towards the center. This can be used for ligand binding or to form a pore or channel for moving small moleculesacross the membrane.

Lodish 4

Kyte-Doolittle plot

Např. http://arbl.cvmbs.colostate.edu/molkit/hydropathy/

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Detergents solubilization of membranes. A major argument for the existence of rafts, as well as the most common test for whether is protein is in a raft, depends on detergent solubilization.

Lodish 4

CMC = critical micelle concentration

RAFTY u živočichů

Triton X-100. Non-denaturing, very low CMC, hard to remove. Rafts components thought to be insoluble at low temperature.

Octylglucoside. Non-denaturing, high CMC, easy to remove. Solubilizes rafts. Expensive (not an industrial detergent).

SDS. Very denaturing. Solublilizes 90% of all membrane proteins (though very hydrophobic ones remain aggregated or insoluble). Imparts uniform negative charge density on proteins, enabling electrophoretic separation based on size alone.

Detergent structures and properties.

Lodish 4

Isolation of “rafts” by flotation in non-ionic detergents.

• Most common method to identify raft components is to extract cells with certain non-ionic detergents, such as Triton-X100 at 4oC, followed by flotation in sucrose gradient. Lubrol extraction may isolate a second type of raft.

• Flotation is important to distinguish from cytoskeletally associated complexes, which are also insoluble in detergent, but pellet incentrifuge.

• Caveat: Proteins that do not associate in vivo can be co-isolated in floating “rafts”, e.g. some mitochondrial proteins end up in the raft fraction. (See Cell 115:377 for latest critique of raft model.)

• Caveat: Detergent extraction causes things to redistribute and collapse into apparent rafts. Such aggregation has been seen even by immunofluorescence microscopy. Lipids and rafts are not immobilized by many conventional fixation procedures, which only immobilize proteins.

• Caveat: Weak interactions with rafts can be dissociated.

| Common tools to disrupt rafts

Cholesterol sequestrationAntibiotics:Filipin | Nystatin | AmphotericinPore-forming agents:Saponin | Digitonin | Streptolysin O

Cholesterol depletionMethyl--cyclodextrin

Inhibition of cholesterol biosynthesisLovastatin

Perturbation of raft stabilityExogenous cholesterol

Exogenous gangliosidesExogenous polyunsaturated fatty acids

Nat Rev MCB 2000 Simons 1:31

The most common approach to disrupting rafts is to deplete or sequester cholesterol, e.g. using methyl-cyclodextrin, which extracts cholesterol from cell membranes. The problem is that almost all physiological membrane events, especially at the plasma membrane, require cholesterol. Depletion of too much cholesterol stops non-raft processes, such as clathrin-mediated endocytosis. Like detergent solubilization, using sensitivity of a process to cholesterol depletion to demonstrate involvement of rafts is now seriously questioned.

11:203

Biophysical techniques have been used to determine clustering of proteins into rafts. Many of these studies have yielded contradictory or confusing results (Cell 115:377). For example, Fluorescence resonance energy transfer (FRET). Sensitive to the 6th power of distance between the two fluorophores. Also depends on degree of spectral overlap, and steric factors. Great revolution is that one or even both fluorophores are GFP variants, rather than conventional small dyes. For instance, pair Cyan fluorescent protein (CFP) with Yellow fluorescent protein (YFP). Clustering of two different GFP-tagged, GPI-anchored proteins can be measured this way. Latest results, and perhaps an emerging consensus, is that rafts are very small (<5 nanometers) and contain an average of ~4 proteins. See Cell 116:577.

65 nm

N terminal domain

C terminal domain

Scaffolding domain

CaveolinsCaveolins and and caveolae; Structure & Functioncaveolae; Structure & Function

Caveolin

• Tumour suppression• Cholesterol transporter/ regulator

intramembrane domain

A. Pol/R. Parton

• Signalling• Muscular dystrophy• Alzheimer’s Disease

Caveolins (1-3)

Caveolae are apparently a specialized, stabilized raft structure. Much literature confuses caveolae and rafts. True caveolae contain caveolin and have a “cave-like” morphology. See also Traffic 4:724

Membrane microdomains and signal transduction. Disruption of caveolin lowers membrane cholesterol content, but might also disrupt segregation into microdomains. Different Ras isoforms associate preferentially with distinct microdomains. Their different signaling properties might

arise from the differential localization of regulators or effectors (X or Y). Nat. cell biology 1 :E35-E37,

Nature Cell Biology 3, 368 - 375

Proposed mechanisms of raft clustering and signaling. (a) Rafts (red) are small at the plasma membrane, containing only a subset of proteins. (b) Raft size is increased by clustering, leading to a new mixture of molecules. This clustering can be triggered (1) at the extracellular side by ligands, antibodies, or lectins, (2) within the membrane by oligomerization, or (3) by cytosolic agents (cytoskeletal elements, adapters, scaffolds). Raft clustering occurs at the plasma membrane as well as intracellularly, e.g., in endosomal lumen. Ligand binding or oligomerization can alter the partitioning of proteins in and out of rafts. Increased raft affinity of a given protein and its activation within rafts (e.g., phosphorylation by Src-family kinases [yellow]) can initiate a cascade of events, leading to further increase of raft size by clustering. J. Clin Invest. 110:597

Simons NRMCB. 1:31. See also Traffic 4:812

Is clustering of rafts involved in signaling? Until recently, one of the best models was the T cell receptor, where activationwas thought to lead to fusion of small rafts and recruitment of many proteins into a large signaling platform at the immunological synapse. Latest data is that rafts may not be involved after all. Nat Cell Biol 6:180. Cell 115:377.

Rostliny a lipidové rafty

Plant Physiology 2005

Summary: Major change in status of raft hypothesis since 2003. 1. Detergent insolubility/flotation and sensitivity to cholesterol depletion are both very non-

specific and are not good evidence that a given protein is in a raft, or even that rafts exist, especially in unperturbed membrane.

2. Sphingolipids are entirely in outer leaflet, while cholesterol is probably enriched in the outer leaflet. Given the high mole % of these in most plasma membrane, almost the entire plasma membrane may be lo phase. If the entire outer leaflet is lo phase, then the word “raft” is the wrong analogy.

3. Signaling events at the outer leaflet are hypothesized to couple to signaling pathways at the inner leaflet, e.g. clustering of GPI-anchored proteins affec.t It is not clear how this trans-bilayer coupling occurs, though interdigitation of the long acyl chains of sphingolipids or role for transmembrane proteins are possibilities.

4. Latest biophysical studies suggest rafts are very small, ~4 proteins. Explains why hard to visualize rafts by light microscopy.

5. Models largely ignore organizing effects of proteins, which are 30-40% of the membrane.

6. My view: There are clearly lateral in-homogeneities of lipids in membranes, but the “raft” hypothesis is probably not the best description. More of a metaphor for a poorly understood, and probably very complex situation. Ann. Rev Cell Dev Bio 20:839

Keith Mostov kriticky o raftech

Složení membrány není homogenní – funkční lipidicko/proteinové

dynamické membránové domény.

Tohle všichni známe z učebnic

….mění selokálněsloženímembránovýchlipidů.

Specific PI lipids are located in specific compartments. Subcellular distribution of the phosphoinositides in (a) yeast and (b) mammalian cells. The kinases and phosphatases acting on the phosphoinositides in the different trafficking pathways are indicated; see this reference for details. The localizations of the different phosphoinositides, deduced by the localisation of specific PIBMs, are color coded for identification. EE, early endosomes; GC, Golgi complex; LE, late endosomes; Ly, lysosomes; MVB, multivesicular body; N, nucleus; V, vacuole. Notice that PI45P2 is mainly localized at the plasma membrane, the 3-phosphorylated phosphoinositides in the endosomal system and PI4P at the GC. The GC in mammalian cells contains also PI45P2, mainly in the stacks, and PI. PI345P3 is localized to specific regions of the PM under specific conditions, often connected with specialized signaling events, such as chemotaxis. Curr Op Cell Bio 14:434. New results: P IP4P in trans-Golgi (Cell 114:299) and PI35P2 in MVB (Dev Cell 5:499).

Základem funkčně special. domén membrán jsou proteinové domény specificky vážící

specifické fosfolipidy.

Recognition of (acidic) phospholipids by proteins

• PH domains: Can recognize PIP2, PI3,4P, PI3,4,5P, etc, with varying degrees of specificity, depending on individual PH domain.

• FYVE domains: Recognize PI3P. Involved in endocytotic pathways.

• ENTH domain: Recognize PI4,5P2, or in some cases PI3,5P2 (Dev. Cell 5:363).

• C1 domains: Recognize diacylglycerol.• Many others. • See Curr Op Cell Biol 13:146 for over-view.

Lipids can be localized to discrete portions of a membrane. In this picture, a PH domain that binds to PI345P3 has been fused to GFP. It is localized to the basolateral plasma membrane of the epithelial cells in this cyst structure, and to extensions that have been induced by treatment with Hepatocyte Growth Factor. How lipids are

constrained to one region of a membrane (in this case part of the plasma membrane), rather than freely diffusing in the plane of the membrane, is not well understood. Tight junctions can prevent lipid diffusion in the outer leaflet of the plasma membrane, but PIP3 is in the inner leaflet.Mol. Biol. Cell, 14:748-763, 2003.

Fosfatidylcholin

PLA1, PLA2, PLC a PLD štěpí na různých místech tentýž

substrát a tedy různě modifikují membránu.

Advantages of recruitment of proteins to lipids

• Many lipid sites can exist, so can target many different proteins to a membrane, without saturating binding sites.

• Lipids can be produced and degraded rapidly by enzymes, time-scale seconds to minutes. Allows the characteristics of a membrane to be rapidly remodeled.

• Lipid binding interactions often low affinity, enables rapid protein equilibration and movement inside cell.

• Lipids are small, so combinations of lipids or lipids and proteins can be used to selectively target proteins to distinct membranes and sites.

Koincidenční mechanismus

dynamiky specifických

membránovýchdomén

Rab GTPázy jako

organizátory membránových

domén.„Zrání“

kompartmentu a konverze Rabů.Podr. v sekreci.