ESCRT-dependent cargo sorting at multivesicular endosomes.

The endosomal sorting complex required for transport (ESCRT) machinery is composed of five multi-subunit protein complexes, which act cooperatively at specialized endosomes to facilitate the movement of specific cargoes from the limiting membrane into vesicles that bud into the endosome lumen. Over the past decade, numerous proteins, lipids, and RNAs have been shown to be incorporated into intralumenal vesicles (ILVs), but the mechanisms by which these unique cargoes are captured are only now becoming better understood. Here, we discuss the potential roles that the ESCRT machinery plays during cargo sorting at multivesicular endosomes (MVEs).

Membrane Transport at an Organelle Interface in the Early Secretory Pathway: Take Your Coat Off and Stay a While: Evolution of the metazoan early secretory pathway.

Most metazoan organisms have evolved a mildly acidified and calcium diminished sorting hub in the early secretory pathway commonly referred to as the Endoplasmic Reticulum-Golgi intermediate compartment (ERGIC). These membranous vesicular-tubular clusters are found tightly juxtaposed to ER subdomains that are competent for the production of COPII-coated transport carriers. In contrast to many unicellular systems, metazoan COPII carriers largely transit just a few hundred nanometers to the ERGIC, prior to COPI-dependent transport on to the cis-Golgi. The mechanisms underlying formation and maintenance of ERGIC membranes are poorly defined. However, recent evidence suggests an important role for Trk-fused gene (TFG) in regulating the integrity of the ER/ERGIC interface. Moreover, in the absence of cytoskeletal elements to scaffold tracks on which COPII carriers might move, TFG appears to promote anterograde cargo transport by locally tethering COPII carriers adjacent to ERGIC membranes. This action, regulated in part by the intrinsically disordered domain of TFG, provides sufficient time for COPII coat disassembly prior to heterotypic membrane fusion and cargo delivery to the ERGIC.

TFG facilitates outer coat disassembly on COPII transport carriers to promote tethering and fusion with ER-Golgi intermediate compartments.

The conserved coat protein complex II (COPII) mediates the initial steps of secretory protein trafficking by assembling onto subdomains of the endoplasmic reticulum (ER) in two layers to generate cargo-laden transport carriers that ultimately fuse with an adjacent ER-Golgi intermediate compartment (ERGIC). Here, we demonstrate that Trk-fused gene (TFG) binds directly to the inner layer of the COPII coat. Specifically, the TFG C terminus interacts with Sec23 through a shared interface with the outer COPII coat and the cargo receptor Tango1/cTAGE5. Our findings indicate that TFG binding to Sec23 outcompetes these other associations in a concentration-dependent manner and ultimately promotes outer coat dissociation. Additionally, we demonstrate that TFG tethers vesicles harboring the inner COPII coat, which contributes to their clustering between the ER and ERGIC in cells. Together, our studies define a mechanism by which COPII transport carriers are retained locally at the ER/ERGIC interface after outer coat disassembly, which is a prerequisite for fusion with ERGIC membranes.

Pathogenic TFG Mutations Underlying Hereditary Spastic Paraplegia Impair Secretory Protein Trafficking and Axon Fasciculation.

Length-dependent axonopathy of the corticospinal tract causes lower limb spasticity and is characteristic of several neurological disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis. Mutations in Trk-fused gene (TFG) have been implicated in both diseases, but the pathomechanisms by which these alterations cause neuropathy remain unclear. Here, we biochemically and genetically define the impact of a mutation within the TFG coiled-coil domain, which underlies early-onset forms of HSP. We find that the TFG (p.R106C) mutation alters compaction of TFG ring complexes, which play a critical role in the export of cargoes from the endoplasmic reticulum (ER). Using CRISPR-mediated genome editing, we engineered human stem cells that express the mutant form of TFG at endogenous levels and identified specific defects in secretion from the ER and axon fasciculation following neuronal differentiation. Together, our data highlight a key role for TFG-mediated protein transport in the pathogenesis of HSP.

Membrane remodeling during embryonic abscission in Caenorhabditis elegans.

Abscission is the final step of cytokinesis and results in the physical separation of two daughter cells. In this study, we conducted a time-resolved series of electron tomographic reconstructions to define the steps required for the first embryonic abscission in Caenorhabditis elegans Our findings indicate that membrane scission occurs on both sides of the midbody ring with random order and that completion of the scission process requires actomyosin-driven membrane remodeling, but not microtubules. Moreover, continuous membrane removal predominates during the late stages of cytokinesis, mediated by both dynamin and the ESCRT (endosomal sorting complex required for transport) machinery. Surprisingly, in the absence of ESCRT function in C. elegans, cytokinetic abscission is delayed but can be completed, suggesting the existence of parallel membrane-reorganizing pathways that cooperatively enable the efficient severing of cytoplasmic connections between dividing daughter cells.

Ist1 regulates ESCRT-III assembly and function during multivesicular endosome biogenesis in Caenorhabditis elegans embryos.

Degradation of most integral membrane proteins is directed by the endosomal sorting complex required for transport (ESCRT) machinery, which selectively targets ubiquitin-modified cargoes into intralumenal vesicles (ILVs) within multivesicular endosomes (MVEs). To better understand the mechanisms underlying ESCRT-mediated formation of ILVs, we exploited the rapid, de novo biogenesis of MVEs during the oocyte-to-embryo transition in C. elegans. In contrast to previous models suggesting that ILVs form individually, we demonstrate that they remain tethered to one another subsequent to internalization, arguing that they bud continuously from stable subdomains. In addition, we show that membrane bending and ILV formation are directed specifically by the ESCRT-III complex in vivo in a manner regulated by Ist1, which promotes ESCRT-III assembly and inhibits the incorporation of upstream ESCRT components into ILVs. Our findings underscore essential actions for ESCRT-III in membrane remodeling, cargo selection, and cargo retention, which act repetitively to maximize the rate of ILV formation.

Burning cellular bridges: Two pathways to the big breakup.

During cytokinetic abscission, the endosomal sorting complex required for transport (ESCRT) proteins are recruited to the midbody and direct the severing of the intercellular bridge. In this issue, Christ et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201507009) demonstrate that two separate but redundant pathways exist to recruit ESCRT-III proteins to the midbody.

Oligoclonal banding in AIDS and hemophilia.

Hypergammaglobulinemia is a consistent finding in patients within the AIDS spectrum and with hemophilia A. Serum samples from patients with these conditions were analyzed for the presence of oligoclonal banding, using a high-resolution serum protein electrophoresis system. The incidence of banding is significantly greater in well homosexuals who are HIV-antibody positive and in patients with pre-AIDS-related complex, AIDS-related complex, AIDS with opportunistic infections, and AIDS with Kaposi's sarcoma than in normal blood donors. The incidence of banding is similar to controls in patients with hemophilia who have received either no blood products, cryoprecipitate only, or limited infusions of factor VIII concentrate. In patients who have received frequent infusions of factor VIII concentrate, the incidence of banding significantly increases. Thirteen of sixty-seven hemophiliac patients developed AIDS or symptoms related to HIV infection independent of their banding pattern. We hypothesize that the bands are not diagnostic of AIDS, but seem to correspond with disease progression, and that they are absent early in the disease, appear later in the course, and may disappear with advanced disease.

Nerve block anesthesia for hair transplantation.

A technique of preliminary nerve block anesthesia for hair transplantation that increases patient comfort by reducing pain and makes hair transplantation a more easily tolerated procedure as compared to local anesthesia alone is described.