Bilayer registry in a multicomponent asymmetric membrane : dependence on lipid composition and chain length

A question of considerable interest to cell membrane biology is whether phase segregated domains across an asymmetric bilayer are strongly correlated with each other and whether phase segregation in one leaflet can induce segregation in the other. We answer both these questions in the affirmative, using an atomistic molecular dynamics simulation to study the equilibrium statistical properties of a 3-component {\em asymmetric} lipid bilayer comprising an unsaturated POPC (palmitoyl-oleoyl-phosphatidyl-choline), a saturated SM (sphingomyelin) and cholesterol with different composition ratios. Our simulations are done by fixing the composition of the upper leaflet to be at the coexistence of the liquid ordered ($l_o$) - liquid disordered ($l_d$) phases, while the composition of the lower leaflet is varied from the phase coexistence regime to the mixed $l_d$ phase, across a first-order phase boundary. In the regime of phase coexistence in each leaflet, we find strong transbilayer correlations of the $l_o$ domains across the two leaflets, resulting in {\it bilayer registry}. This transbilayer correlation depends sensitively upon the chain length of the participating lipids and possibly other features of lipid chemistry, such as degree of saturation. We find that the $l_o$ domains in the upper leaflet can {\em induce} phase segregation in the lower leaflet, when the latter is nominally in the mixed ($l_d$) phase.Comment: 6 figure

Rafts: scale-dependent, active lipid organization at the cell surface

Rafts have been conceptualized as lateral heterogeneities in the organization of cholesterol and sphingolipids, endowed with sorting and signaling functions. In this review we critically examine evidence for the main tenet of the 'raft hypothesis', namely lipid-dependent segregation of specific membrane components in the plasma membrane. We suggest that conventional approaches to studying raft organization wherein membranes are treated as passive, thermally equilibrated systems are unlikely to provide an adequate framework to understand the mechanisms of raft-organization in vivo. An emerging view of raft organization is that it is spatio-temporally regulated at different scales by the cell. This argues that rafts must be defined by simultaneous observation of components involved in particular functions. Recent evidence from the study of glycosylphosphatidyl inositol-anchored proteins, a common raft-marker, supports this picture in which larger scale, more stable rafts are induced from preexisting small-scale lipid-dependent structures actively maintained by cellular processes

ARF1 is directly involved in dynamin-independent endocytosis

Endocytosis of glycosylphosphatidyl inositol (GPI)-anchored proteins (GPI-APs) and the fluid phase takes place primarily through a dynamin- and clathrin-independent, Cdc42-regulated pinocytic mechanism. This mechanism is mediated by primary carriers called clathrin-independent carriers (CLICs), which fuse to form tubular early endocytic compartments called GPI-AP enriched endosomal compartments (GEECs). Here, we show that reduction in activity or levels of ARF1 specifically inhibits GPI-AP and fluid-phase endocytosis without affecting other clathrin-dependent or independent endocytic pathways. ARF1 is activated at distinct sites on the plasma membrane, and by the recruitment of RhoGAP domain-containing protein, ARHGAP10, to the plasma membrane, modulates cell-surface Cdc42 dynamics. This results in the coupling of ARF1 and Cdc42 activity to regulate endocytosis at the plasma membrane. These findings provide a molecular basis for a crosstalk of endocytosis with secretion by the sharing of a key regulator of secretory traffic, ARF1

Phosphorylation of nephrin induces phase separated domains that move through actomyosin contraction

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Kim, S., Kalappurakkal, J. M., Mayor, S., & Rosen, M. K. Phosphorylation of nephrin induces phase separated domains that move through actomyosin contraction. Molecular Biology of the Cell, 30(24), (2019): 2996–3012, doi:10.1091/mbc.E18-12-0823.The plasma membrane of eukaryotic cells is organized into lipid and protein microdomains, whose assembly mechanisms and functions are incompletely understood. We demonstrate that proteins in the nephrin/Nck/N-WASP actin-regulatory pathway cluster into micron-scale domains at the basal plasma membrane upon triggered phosphorylation of transmembrane protein nephrin. The domains are persistent but readily exchange components with their surroundings, and their formation is dependent on the number of Nck SH3 domains, suggesting they are phase separated polymers assembled through multivalent interactions among the three proteins. The domains form independent of the actin cytoskeleton, but acto-myosin contractility induces their rapid lateral movement. Nephrin phosphorylation induces larger clusters at the cell periphery, which are associated with extensive actin assembly and dense filopodia. Our studies illustrate how multivalent interactions between proteins at the plasma membrane can produce micron-scale organization of signaling molecules, and how the resulting clusters can both respond to and control the actin cytoskeleton.We thank Hongtao Yu (University of Texas Southwestern Medical Center [UTSW]) for providing the HeLa cell line used in this work; Dan Billadeau and Timothy Gomez (Mayo Clinic) for providing antibodies; Nico Stuurman (University of California, San Francisco) for assistance with STORM imaging; Kate Luby-Phelps and Abhijit Bugde (UTSW Live Cell Imaging Core Facility) for their assistance in epifluorescence and spinning disk confocal experiments; Sudeep Banjade for advice on designing the S3, S2, S1 constructs; Khuloud Jaqaman (UTSW) for advice on cluster motility analysis; Salman Banani and Jonathan Ditlev (UTSW) for critical reading of the manuscript; and members of the Rosen lab and participants in the MBL/HHMI Summer Institutes for advice and helpful discussions. This work was supported by a Howard Hughes Medical Institute Collaborative Innovation Award; the Welch Foundation (I-1544 to M.K.R.); a J.C. Bose Fellowship from the Department of Science and Technology, government of India (to S.M.); a Margadarshi Fellowship from the Wellcome Trust—Department of Biotechnology, India Alliance (IA/M/15/1/502018 to S.M.). Research in the Rosen lab is supported by the Howard Hughes Medical Institute

A role for the Hsp90 molecular chaperone family in antigen presentation to T lymphocytes via major histocompatibility complex class II molecules

The heat shock protein (HSP) Hsp90 is known to chaperone cytosolic peptides for MHC class I (MHCI)-restricted antigen presentation to T lymphocytes. We now demonstrate a role for Hsp90 activity in presentation of antigens on MHCII. Treatment of mouse antigen-presenting cells (APC) with the pharmacological Hsp90 inhibitor, geldanamycin, inhibited MHCII-mediated presentation of endocytosed and cytosolic proteins as well as synthetic peptides to specific T cells. Ectopic expression of human Hsp90 in APC enhanced MHCII-mediated antigen presentation. Further, pharmacological Hsp90 inhibition reduced, while retroviral Hsp90 overexpression enhanced, the levels of stable compact MHCII heterodimers correlating with the antigen presentation phenotype. Pharmacological inhibition of Hsp90 activity in IFN-γ-treated APC resulted in severe abrogation of MHCII-restricted presentation of cytosolic antigen, but only partially inhibited exogenous antigen presentation. Our data suggest a major role for Hsp90 activity in MHCII-mediated antigen presentation pathways, and implicate IFN-γ-inducible Hsp90-independent mechanisms

deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster

Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function

Stratification relieves constraints from steric hindrance in the generation of compact acto-myosin asters at the membrane cortex

Recent in-vivo studies have revealed that several membrane proteins are driven to form nanoclusters by active contractile flows arising from F-actin and myosin at the cortex. The mechanism of clustering was shown to be arising from the dynamic patterning of transient contractile platforms (asters) generated by actin and myosin. Myosin-II, which assemble as minifilaments consisting of tens of myosin heads, are rather bulky structures and hence a concern could be that steric considerations might obstruct the emergence of nanoclustering. Here, using coarse-grained, agent-based simulations that respect the size of constituents, we find that in the presence of steric hindrance, the patterns exhibited by actomyosin in two dimensions, do not resemble the steady state patterns observed in our in-vitro reconstitution of actomyosin on a supported bilayer. We then perform simulations in a thin rectangular slab, allowing the separation of a layer of actin filaments from those of myosin-II minifilaments. This recapitulates the observed features of in-vitro patterning. Using super resolution microscopy, we find direct evidence for stratification in our in-vitro system. Our study suggests the possibility that molecular stratification may be an important organising feature of the cortical cytoskeleton in-vivo

HIV-1 Nef induces a Rab11-dependent routing of endocytosed immune costimulatory proteins CD80 and CD86 to the Golgi

The Nef protein of HIV-1 removes the immune costimulatory proteins CD80 and CD86 from the cell surface by a unique clathrin- and dynamin-independent, actin-based endocytic pathway that deploys coupled activation of c-src and Rac. In this study, we show that, similar to major histocompatibility complex class I (MHCI), Nef subsequently reroutes CD80 and CD86 to the Golgi region. However, not only are CD80/CD86 internalized by a different mechanism from MHCI but also the vesicular pathway of Golgi delivery for CD80/CD86 is distinct from that employed for MHCI. While MHCI passes through early endosomal and sorting compartments marked by Rab5/early embryonic antigen 1 and ADP ribosylation factor 6, respectively, CD80 and CD86 enter endocytic vesicles that do not acquire conventional early endosomal markers but remain accessible to fluid probes. Rather than being delivered to preexisting Rab11-positive recycling compartments, these vesicles recruit Rab11 de novo. Rab11 activity is also necessary for the delivery of CD80/CD86 in these transitional vesicles to the Golgi region. These data reveal an unusual pathway of endocytic vesicular traffic to the Golgi and its recruitment in a viral immune evasion strategy

HIV-1 Nef promotes endocytosis of cell surface MHC class II molecules via a constitutive pathway

HIV-1 Nef has been reported to disrupt MHC class II (MHCII)-mediated Ag presentation by a dual strategy that comprises a reduction in cell surface levels of peptide-loaded mature MHCII molecules and a up-regulation of immature MHCII molecules. We show that Nef achieves relocation of MHCII away from the cell surface in monocytic cells by both delaying its transport to the cell surface and by accelerating endocytic removal of cell surface MHCII to a lysosomal compartment. Nef-induced MHCII endocytosis is cholesterol-sensitive but clathrin- and dynamin-independent. Internalized MHCII molecules traverse the early endosomal system and colocalize with pinocytic cargo before reaching lysosomes. Nef-triggered MHCII endocytosis requires Rab5 activity and lyst function, whereas lysosomal trafficking of internalized MHCII molecules requires Rab7 activity. We further show that a similar pathway can remove peptide-MHCII complexes from the surface of monocytic cells not expressing Nef. Our data suggest that Nef uses mechanisms involved in normal MHCII recycling and turnover to mediate the delivery of cell surface MHCII to a lysosomal destination. Thus, Nef-mediated endocytosis of MHCII provides a novel perspective on the regulation of normal MHCII trafficking

Efficient presentation of both cytosolic and endogenous transmembrane protein antigens on MHC class II is dependent on cytoplasmic proteolysis

Peptides from extracellular proteins presented on MHC class II are mostly generated and loaded in endolysosomal compartments, but the major pathways responsible for loading peptides from APC-endogenous sources on MHC class II are as yet unclear. In this study, we show that MHC class II molecules present peptides from proteins such as OVA or conalbumin introduced into the cytoplasm by hyperosmotic pinosome lysis, with efficiencies comparable to their presentation via extracellular fluid-phase endocytosis. This cytosolic presentation pathway is sensitive to proteasomal inhibitors, whereas the presentation of exogenous Ags taken up by endocytosis is not. Inhibitors of nonproteasomal cytosolic proteases can also inhibit MHC class II-restricted presentation of cytosolically delivered protein, without inhibiting MHC class I-restricted presentation from the same protein. Cytosolic processing of a soluble fusion protein containing the peptide epitope I-Eα 52-68 yields an epitope that is similar to the one generated during constitutive presentation of I-Eα as an endogenous transmembrane protein, but is subtly different from the one generated in the exogenous pathway. Constitutive MHC class II-mediated presentation of the endogenous transmembrane protein I-Eα is also specifically inhibited over time by inhibitors of cytosolic proteolysis. Thus, Ag processing in the cytoplasm appears to be essential for the efficient presentation of endogenous proteins, even transmembrane ones, on MHC class II, and the proteolytic pathways involved may differ from those used for MHC class I-mediated presentation