Less well defined extracellular membranous containers

Question 1

Cell-in-cell structures

Answer 1

• rare events
• internalization of a cell of an organism in other cells (entosis)
• temporary, being on the move through a cell (emperipolesis) or permanent (?) or as a part of a death pathway of the engulfed cell
• target cell is non-apoptotic and sometimes even divides
• host cells non-neoplastic of various cell types, but may be also neoplastic
• target cell is often of haematopoetic lineage

Question 2

Extrazellular Vesicles

Answer 2

• Exosomes
• Multivesicles

Question 3

Multivesicles

Answer 3

• different names (microvesicles, ectosomes, shedding vesicles, Microparticles), observed under apoptotic and non-ptotic conditions
• existence of a non-blebbing non-apoptotic process with secretory function as source of TVs (ectocytosis) is not well established; mechanism, occurrence and function is not well understood; supposed to play role in intracellular communication
• best evidence exists for an TSG101/ESCRT III dependent process which may be active in cells at low basal rate continuously
• Ectosomes are probably linked with clearance of “attacking” complexes like complement, pore forming toxins or repair of small (< 100 nm) PM-ruptures, processes which are also reported to be ESCRT dependent

Question 4

Membrane blebbing

Answer 4

processes with outward budding of PM membranes
- reversible nonapoptotic blebs, probably involved in cell migration through ECM
- blebbing in apoptotic cells (relative large areas) resulting in the pinch-off of these areas; remnants of blebs are believed to be one source of multiversicles (MV)

Question 5

Ejectosome

Answer 5

• structure observed during ejection of intracellular pathogens (e.g. Mycobacterium)
• actin-based mechanism perhaps descended from plasma membrane repair mechanisms
• induction needs secreted pathogen factors

1) protrusion
2) ingression
3) resealing

Question 6

Distribution of membrane lipids in opistokont cells

Answer 6

• Bulk synthesis of lipids in the ER, esp also at ER-Mito contact sites
• Mito synthesizes 45 % of its lipid content
• Golgi is involved in biosynthesis of some lipids

Question 7

Transport of lipids between compartments - Principles

Answer 7

• most compartments do not synthesize all their amphiphilic membrane lipids themselves; transport must traverse an aqueous barrier formed by one of the plasmatic phases of a cell or a hydrophobic barrier

Mechanisms:
- spontaneous exchange only fast for short or single-chain lipids (e.g. minutes for lysophosphatidylcholin)
- lipid transport using the membranous transport containers involved in protein transport
- lipid transfer proteins; also important for formation of lipid droplets
a) via diffusion
b) via membrane contact sites
- lipid exchange between leaflets of the same membrane occurs usually via flippases (ATP-dependent and ATP-independent)

Question 8

Formation of asymmetry in lipid composition along the secretory pathway

Answer 8

In the ER, non-specific transbilayer equilibration of phospholipids has been demonstrated, and the membrane exhibits a nearly symmetric lipid distribution between bilayer leaflets. In the Golgi, P4 ATPases translocate PS and PE to the cytosolic face. Sphingomyelin (SM) is produced from ceramide (Cer) on the luminal side. Asymmetry is generated by the specific transport of PS and PE and lack of transport of SM. in SM synthesis, PC is converted to diacylglycerol (DAG), which freely equilibrates across bilayers and can serve as a substrate for cholinephosphotransferase isozyme, the product of which is PC.

Question 9

Maintenance of asymmetry in lipid composition along the secretory pathway

Answer 9

At the plasma membrane, P4 ATPases transport PS and PE to the cytosolic face. This homeostatic distribution can be disrupted by activation of scramblase and/or inhibition of the P4 ATPases.
Within endosomes, fluorescently labeled PC, SM and glycosphingolipids (GSLs) were shown to be restricted to the luminal leaflet.

Question 10

Vesicular transport and lipid homeostasis

Answer 10

• vesicular transport could be used for “bulk” lipid transport
  -> two forms:
  1) almost protein free vesicles ???? (SNAREs are always needed!)
  2) lipid supply coupled to protein sorting
• vesicular transport must maintain the lipid disequilibrium between compartments
  -> existence of an extensive lipid sorting during vesicle formation?
• lipid transfer (e.g. between ER and PM) is faster (t1/2 2-5 min) than vesicle traffic would allow and is only marginally affected by disruption of the secretory pathway

Question 11

Membrane contact sites

Answer 11

A lipid-transport network based on the ER; membrane contact sites. E.g. PE is synthesized in the mitochondrial matrix, and might move to the plasma membrane by a non-vesicular route.

Question 12

Membrane contact sites and lipid synthesis

Answer 12

interplay between vesicular and non-vesicular lipid transfer broadens the possibility of pathways for glycolipid synthesis

Question 13

Lipid exchange at ER-MT CS

Answer 13

During biosynthesis of two of the cell’s most abundant phospholipids, PC and PE, PS is first made on the ER, but it must be translocated to the outer mitochondrial membrane (OMM) and then transferred to the inner mitochondrial membrane (IMM), where it is converted to PE. To make PC, the PE precursor must then be translocated from the OMM to the ER, where it is modified to make PC. There must also be a mechanism by which PC is translocated back from the ER to the OMM, as mitochondria also contain PC.

Question 14

Lipid droplets

Answer 14

• principle site of neural lipid storage present in most cells (fungi, metazoan, viridiplanta)
• Dynamic entity:
  -> genesis at the ER; usually budding to free LD (initial LD, 0,3-0,6 µm)
  -> growth up to 1 … 20 µm depending on cell type (expanding LD); models:
• by lipogenesis at the ER and transfer to the still connected LD
• by lipogenesis at the ER and transfer to the pinched off LD via lipid exchange proteins
• by lipogenesis at the ER and budding of small new synthesized LD and their fusion to pinched off old LD
• by lipogenesis at the ER and budding of small new synthesized LD which transfer their TAG to a pinched off old LD via lipid exchange proteins
• by lipogenesis at the pinched off LD
• breakdown (lipolysis)
  -> first large LD fragment (supported by ARF and COPI ?)
  -> recruitment of lipases and/or Rab18, which leads to contact to ER

Question 15

Lipid droplets - composition

Answer 15

different types of peripheral membrane proteins
- enzyme complexes fro neutral fat synthesis
- LPCAT for synthesis of phospholipids
- PAT/Plin proteins (e.g. ADRP) protecting (coating) against access of lipases
- structural proteins of unknown functions (seipin, FIT)
- SNAREs (SNAP23, syntaxin 5, VAMP4) which mediate fusion of LD
- proteins with hydrophobic hairpin-loops are targeted via ER; all other come direct from the cytosol

LD of one cell can have different metabolic activity and hence different protein composition

Question 16

Formation of LD

Answer 16

Formation occurs at the ER; there may be a large amount of LD that stay connected with the ER; contact-sites are regulated by Rab18 ?

Question 17

Lipid droplet - organelle contacts

Answer 17

Lipid droplets interact with nearly all organelles in the cell.
Although the organelle contacts are depicted as distinct and spatially separate, it is likely that individual lipid droplets participate in contacts with multiple organelles simultaneously.

Question 18

What defines a compartment of the secretory pathway and how are structures maintained under conditions of constant protein and lipid flow?

Answer 18

Static:
- sets of integral membrane proteins (enzymes and structural proteins) (positively) or their exclusion (negatively) define compartments and their subdomains (e.g. ER)
- maintenance is guaranteed by retention/retrieval mechanisms

Dynamic:
- scaffold of proteins organized by Rabs (or other small GTPases) and combinatorial interaction between Rabs, membrane proteins and lipids define the different sorting platforms (membrane domains) of an compartment (TGN, endosomal/lysosomal compartments);
- scaffold is basis for delivery from, but often also reception and integration of the correct vesicles into the target membrane domain via SNARE-pairing

Question 19

Phagocytosis-like cell engulfment by planctomycete bacterium

Answer 19

Ca. Hab amorphum
- large (5x10 µm)
- amoeboid movement
- contains genes for peptidoglycan synthesis
- contains actin-like proteins related to actin-like genes in Lokiarchea

Question 20

Diversification of compartments of the secretory pathway is still occurring

Answer 20

Human Cathrin isoform CHC22 defines a specific coat used for transport of GLUT4 to the Glut4 storage compartment (GSC).