MEMBRÁNY Lipidové dvojvrstvy Plasmalema & co.

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Věra Holubová
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1 MEMBRÁNY Lipidové dvojvrstvy Plasmalema & co.

2 Visí na stěně HECHTOVA VLÁKNA

3 SPOUSTA RŮZNÝCH MEMBRÁN V BUŇCE

4 Základní otázka buněčné biologie JAK SE UDRŽÍ RŮZNÉ SLOŽENÍ MEMBRÁN?

5 Kompartmentace metabolismu lipidů v rostlinné buňce

6 U rostlin spolupracují dvě dráhy syntézy membránových lipidů I. Plastidová ( prokaryotická ) a II. ER ( eukaryotická užívá také mastných kyselin z plastidů) při tvorbě komplexní struktury plastidových membrán.

7 JAK VZNIKAJÍ?

8 K syntéze membr. lipidů dochází na vnější vrstvě ER membrány 1. Acyltransferázy připojují postupně dvě mastné kyseliny ke glycerolfosfátu = vzniká kyselina fosfatidová (PA) už zůstává ve vnější membráně 2. Konečně je připojena specifická hydrofilní skupina: cholin (vzniká fosdatidylcholin=lecithin), nebo ethanolamin(vzniká ), nebo serin ( ) nebo inositol (fosfatidylinositol).

9 Symetrie/asymetrie membrán je udržována scramblázami či flipázami (podobné ABC transporterům), které přenášejí fosfolipidy do vnitřníčásti lipidové dvojvrstvy. Anglické i české názvosloví není dobře ustáleno.

10 Skrambláza/Flipáza působí asymetrii/symetrii v membránové dvouvrstvě

11 NA PM ŽIVOČICHŮ JE DOLOŽENA ASYMETRIE PŮSOBENÁ PŘENOSEM FOSFATIDYLSERINU A FOSFATIDYLETHANOLAMINU DO VNITŘNÍ MEMBRÁNY = SMĚREM K CYTOPLASMĚ.

12 Stěhování membránových lipidů hydrofilním prostředím buňky je pak umožňováno aktivitou řady lipidy přenášejících bílkovin (lipid transfer protein LTP).

15 cis = vazba působí deformaci

16 Fosfatidylcholin

17 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

18 STEROLY

19 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.

21 V oleosomech triglyceridy jako zásobní! lipid.

23 Pohybové možnosti membránových lipidů

25 Fluid Bilayer View of Membrane Structure Lodish 4

26 Topologie membránových bílkovin

27 Single-spanning Membrane Protein: 1 alpha Helix Délka transmembránového úseku je typická pro jednotlivé kompartmenty a od ER k PM se zvětšuje! (tj. zvětšuje se tloušťka dvojvrstvy!) Lodish 4

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.

28 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 molecules across the membrane. Lodish 4

29 Kyte-Doolittle plot - + Predikce hydrofobních TM úseků membrány. Např.

30 HYDROFOBNÍ MODIFIKACE PERIFERNÍCH MEMBR. BÍLKOVIN

31 Detergents solubilization of membranes. A major argument for the existence of rafts, as well as the most common test for whether protein is in a raft, depends on detergent solubilization. RAFTY CMC = critical micelle concentration Lodish 4

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

32 Detergent structures and properties. 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). Lodish 4 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.

33 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 4 o C, 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 in centrifuge. Caveat: Proteins that do not associate in vivo can be coisolated 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.

Membrane microdomains and signal transduction. Disruption of caveolin lowers membrane cholesterol content, but might also disrupt segregation into microdomains.

34 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,

SHLUKOVÁNÍ MIKRORAFTŮ PO STIMULACI EXTRA/INTRA - celulární Proposed mechanisms of raft clustering and signaling. (a) Rafts (red) are small at the plasma membrane, containing only a subset of proteins.

35 SHLUKOVÁNÍ MIKRORAFTŮ PO STIMULACI EXTRA/INTRA - celulární 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

36 Is clustering of rafts involved in signaling? Until recently, one of the best models was the T cell receptor, where activation was 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. Simons NRMCB. 1:31. See also Traffic 4:812

37 Plant Physiology 2005

38 TM=total membr.

42 Keith Mostov kriticky o raftech Summary: Major change in status of raft hypothesis since Detergent insolubility/flotation and sensitivity to cholesterol depletion are both very nonspecific 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 transbilayer 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

44 Metoda základ opět FLOTACE

45 Tab I asi 30 membr. transporterů

47 CELKEM V DRM 145 BÍLKOVIN.

48 LOKÁLNÍ MODIFIKACE SLOŽENÍ LIPIDŮ MEMBRÁNY USNADŇUJE TVORBU A FŮZI VÁČKŮ!

49 KONCEPT TVAROVÉ ROZMANITOSTI LIPIDOVÝCH AGREGÁTŮ Shape-structure concept of lipid polymorphism Traffic 1:605

50 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

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