Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin mediated endocytosis

SUMMARYAs the principal internalization mechanism in mammalian cells, clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of disrupted cholesterol synthesis, sterol specificity, mechanisms involved, and relevance to disease pathology. Using genome-edited cell lines, we demonstrate that inhibition of post-squalene cholesterol biosynthesis as observed in inborn errors of cholesterol metabolism, results in striking immobilization of CME and impaired transferrin uptake. Imaging of membrane bending dynamics and CME pit ultrastructure revealed prolonged clathrin pit lifetimes and accumulation of shallow clathrin-coated structures that scaled with diminishing sterol abundance. Moreover, fibroblasts derived from Smith-Lemli-Opitz syndrome subjects displayed reduced CME function. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest reduced CME contributes to cellular phenotypes observed within disorders of cholesterol metabolism.

A genome-wide almanac of co-essential modules assigns function to uncharacterized genes

SUMMARYA central remaining question in the post-genomic era is how genes interact to form biological pathways. Measurements of gene dependency across hundreds of cell lines have been used to cluster genes into ‘co-essential’ pathways, but this approach has been limited by ubiquitous false positives. Here, we develop a statistical method that enables robust identification of gene co-essentiality and yields a genome-wide set of functional modules. This almanac recapitulates diverse pathways and protein complexes and predicts the functions of 102 uncharacterized genes. Validating top predictions, we show that TMEM189 encodes plasmanylethanolamine desaturase, the long-sought key enzyme for plasmalogen synthesis. We also show that C15orf57 binds the AP2 complex, localizes to clathrin-coated pits, and enables efficient transferrin uptake. Finally, we provide an interactive web tool for the community to explore the results (coessentiality.net). Our results establish co-essentiality profiling as a powerful resource for biological pathway identification and discovery of novel gene functions.

ERM proteins: The missing actin linkers in clathrin-mediated endocytosis

Canonical clathrin-coated pits (CCPs) are nucleated by the coordinated arrival of clathrin triskelia and AP2 adaptor proteins at phosphatidylinositol 4,5-bisphosphate (PIP2) enriched plasma membrane domains (1, 2). Subsequent propagation of the clathrin lattice helps to deform the membrane into a sharply curved pit (3). A large proportion of the initiated pits fall apart as abortive endocytic events within about 20 s, possibly due to insufficient cargo capture (4). Successful clathrin-mediated endocytosis (CME) is concluded when a clathrin-coated vesicle is released into the cell by dynamin-mediated fission of the CCP membrane neck (5, 6). A vast array of accessory proteins important for successful CME has been identified. Among these is actin, which has been shown to be required for the maturation and internalization of a subset of CCPs in human cells (7, 8). Actin dependency during CME correlates with elevated membrane tension, and CCP maturation requires actin polymerization during mitosis, in microvilli at the apical surface of polarized cells and in cells upon global mechanical stretching or hypotonic treatment (9–11). The Arp2/3 complex is thought to trigger an acute actin burst coinciding with CCP internalization, but how actin is recruited to CCPs in the first place is not known (12, 13). Here we show that the ERM (ezrin, radixin, moesin) protein family of membrane-actin linkers associates with CCPs, and that functional perturbation of ERM proteins impedes CCP maturation and reduces the rate of transferrin uptake. By total internal reflection fluorescence (TIRF) microscopy and unbiased object detection and tracking, we show that ezrin localizes to nascent CCPs and that these pits subsequently recruit actin. Based on these data, we propose a model in which activated ERM proteins recruit the initial actin filament during CME.

Flavivirus internalization is regulated by a size-dependent endocytic pathway

Flaviviruses enter host cells through the process of clathrin-mediated endocytosis, and the spectrum of host factors required for this process are incompletely understood. Here we found that lymphocyte antigen 6 locus E (LY6E) promotes the internalization of multiple flaviviruses, including West Nile virus, Zika virus, and dengue virus. Perhaps surprisingly, LY6E is dispensable for the internalization of the endogenous cargo transferrin, which is also dependent on clathrin-mediated endocytosis for uptake. Since viruses are substantially larger than transferrin, we reasoned that LY6E may be required for uptake of larger cargoes and tested this using transferrin-coated beads of similar size as flaviviruses. LY6E was indeed required for the internalization of transferrin-coated beads, suggesting that LY6E is selectively required for large cargo. Cell biological studies found that LY6E forms tubules upon viral infection and bead internalization, and we found that tubule formation was dependent on RNASEK, which is also required for flavivirus internalization, but not transferrin uptake. Indeed, we found that RNASEK is also required for the internalization of transferrin-coated beads, suggesting it functions upstream of LY6E. These LY6E tubules resembled microtubules, and we found that microtubule assembly was required for their formation and flavivirus uptake. Since microtubule end-binding proteins link microtubules to downstream activities, we screened the three end-binding proteins and found that EB3 promotes virus uptake and LY6E tubularization. Taken together, these results highlight a specialized pathway required for the uptake of large clathrin-dependent endocytosis cargoes, including flaviviruses.

Structural inhibition of dynamin-mediated membrane fission by endophilin

Dynamin, which mediates membrane fission during endocytosis, binds endophilin and other members of the Bin-Amphiphysin-Rvs (BAR) protein family. How endophilin influences endocytic membrane fission is still unclear. Here, we show that dynamin-mediated membrane fission is potently inhibited in vitro when an excess of endophilin co-assembles with dynamin around membrane tubules. We further show by electron microscopy that endophilin intercalates between turns of the dynamin helix and impairs fission by preventing trans interactions between dynamin rungs that are thought to play critical roles in membrane constriction. In living cells, overexpression of endophilin delayed both fission and transferrin uptake. Together, our observations suggest that while endophilin helps shape endocytic tubules and recruit dynamin to endocytic sites, it can also block membrane fission when present in excess by inhibiting inter-dynamin interactions. The sequence of recruitment and the relative stoichiometry of the two proteins may be critical to regulated endocytic fission.

Abstract 385: Cardiac Myosin Binding Protein-C Mutants Disrupt Ubiquitin Proteasome System and Hsc70 Functions

The most commonly mutated gene in hypertrophic cardiomyopathy (HCM) is cardiac myosin binding protein C (MYBPC3). Over 90% of MYBPC3 mutations are nonsense, but whether these mutations manifest in loss- or gain-of-function is unresolved. Evidence suggests MYBPC3 mutants impact protein quality control mechanisms. The objective of this study was to evaluate interactions of MYBPC3 with proteostatic systems and test the hypothesis that these interactions affect protein homeostasis in cardiomyocytes. WT and mutant MYBPC3 constructs were expressed in neonatal rat ventricular myocytes (NRVMs) via adenovirus. Mutant MYBPC3 induced ubiquitin proteasome system reporter GFPu accumulation (fold increase in GFPu-positive cells vs control: WT 138±14.0%, mutant 198±27.2%, mean±SEM, p<0.05 vs control and WT), indicating proteasome dysfunction. Affinity purification/mass spectrometry identified molecular chaperones Hsp70 and Hsc70 as prominent interactors with MYBPC3. We observed MYBPC3 degradation by cycloheximide chase in response to Hsc70 siRNA knockdown or pharmacological treatment with Hsp70 activator YM-1. Hsc70 knockdown slowed degradation of WT and mutant MYBPC3 (WT control t ½ =5.47±0.70 hr, WT Hsc70 knockdown t ½ =13.5±1.62 hr; mutant control t ½ =3.42±0.61 hr, mutant Hsc70 knockdown t ½ =9.87±0.95 hr), while YM-1 treatment accelerated degradation (WT DMSO t ½ =10.2±3.28 hr, WT YM-1 t ½ =3.16±0.61 hr; mutant DMSO t ½ =11.7±2.67 hr, mutant YM-1 t ½ =1.37±0.16 hr). We then evaluated whether transferrin uptake via clathrin mediated endocytosis, a critical Hsc70-dependent activity, was affected by mutant MYBPC3. Transferrin uptake was significantly decreased in NRVMs expressing mutant MYBPC3 compared to WT and untreated controls (transferrin-positive cells: control 22.93±3.34%, WT 17.47±0.70%, mutant 9.30±1.63%, mean±SEM, p<0.05 vs control and WT). In conclusion, we have demonstrated that Hsp70 chaperones interact with MYBPC3 in cardiomyocytes and affect MYBPC3 degradation, suggesting MYBPC3 is a client of Hsp70 and Hsc70. Additionally, expression of mutant MYBPC3 causes ubiquitin proteasome impairment and interferes with normal Hsc70 function. These results support our hypothesis that mutant MYBPC3 affects protein homeostasis in HCM.

Microtubule plus end–associated CLIP-170 initiates HSV-1 retrograde transport in primary human cells

Dynamic microtubules (MTs) continuously explore the intracellular environment and, through specialized plus end–tracking proteins (+TIPs), engage a variety of targets. However, the nature of cargoes that require +TIP-mediated capture for their movement on MTs remains poorly understood. Using RNA interference and dominant-negative approaches, combined with live cell imaging, we show that herpes simplex virus particles that have entered primary human cells exploit a +TIP complex comprising end-binding protein 1 (EB1), cytoplasmic linker protein 170 (CLIP-170), and dynactin-1 (DCTN1) to initiate retrograde transport. Depletion of these +TIPs completely blocked post-entry long-range transport of virus particles and suppressed infection ∼5,000-fold, whereas transferrin uptake, early endosome organization, and dynein-dependent movement of lysosomes and mitochondria remained unaffected. These findings provide the first insights into the earliest stages of viral engagement of MTs through specific +TIPs, akin to receptors, with therapeutic implications, and identify herpesvirus particles as one of a very limited number of cargoes absolutely dependent on CLIP-170–mediated capture to initiate transport in primary human cells.

A New AHI-1-DNM2-BCR-ABL Complex Regulates Endocytosis Processes in Chronic Myeloid Leukemia

Tyrosine kinase inhibitors (TKIs) have been introduced into clinical practice with remarkable effects on chronic phase CML. However, early relapses, acquired drug resistance, and persistence of leukemic stem cells remain problematic. Improved treatment approaches to target other key molecular elements active in CML stem/progenitor cells are needed. One candidate is AHI-1 (Abelson helper integration site-1), an oncogene that is highly deregulated in CML stem cells. It harbors two key domains, SH3 and WD40-repeat, which are known important mediators of protein-protein interactions. The AHI-1-mediated protein complex containing BCR-ABL and JAK2 has been shown to mediate transforming activity and TKI-response/resistance of CML stem/progenitor cells. We have recently identified Dynamin-2 (DNM2) as another AHI-1 interacting protein using the AHI-1 SH3 domain as protein ‘bait’ in immunoprecipitation/mass spectrometry. DNM2, a large GTPase, is mainly involved in the trafficking processes such as endocytosis, and is activated through tyrosine phosphorylation. Its role in the mediation of CML stem cell functions is unknown. We have now demonstrated that transcript levels of DNM2 are significantly increased in pre-treatment CD34+ stem/progenitor cells from CML patients who were classified retrospectively, after IM therapy, as IM-responders (n=11) and IM-nonresponders (n=15) as compared to CD34+ normal bone marrow cells (n=7, p=0.013 and 0.037). In addition, DNM2 is more highly expressed in CML stem cells (CD34+CD38-) and progenitor cells (CD34+CD38+) than more mature cells (CD34- , 2-fold). Co-immunopreciptation with mutant forms of AHI-1 and DNM2 (HA-AHI-1, HA-AHI-1 SH3Δ, Myc-DNM2 and Myc-DNM2 PRDΔ) in 293T cells indicated that the PRD domain of DNM2 is mainly responsible for the interaction between AHI-1 and DMN2. Co-localization analysis using confocal microscopy further demonstrated that the interaction between full-length AHI-1 and DNM2 occurs in a “punctate dot” pattern throughout the whole cytoplasm; in contrast, the co-localization signals were significantly disrupted in cells co-transfected with AHI-1 and DNM2 mutants. Interestingly, in AHI-1 SH3D mutant cells, AHI-1was located in the nucleus, suggesting that the SH3 domain of AHI-1 is required for AHI-1 cytoplasmic retention. Moreover, AHI-1 was observed to co-localize with EEA-1 (early endosome marker) and LAMP-1 (late endosome marker) in cells co-transfected with full-length AHI-1 and DNM2, but not in AHI-1 and DNM2 mutant cells. Increased transferrin signals were also demonstrated in AHI-1 and DNM2 co-transfected cells compared to their mutant cells, using transferrin uptake assays, suggesting that the interaction between DNM2 and AHI-1 indeed increases the kinetics and efficiency of endocytosis. These results were further confirmed in BCR-ABL-transduced and BCR-ABL/AHI-1 co-transduced hematopoietic cells that are relatively resistant to TKI-induced apoptosis. Particularly, the co-localization signals between DNM2 and AHI-1 were stronger in BCR-ABL/AHI-1 co-transduced cells, and the transferrin uptake was also more efficient as compared to BCR-ABL-transduced cells. On the other hand, transferrin uptake was reduced in CML cells with knockdown of DNM2 and these cells also demonstrated reduced proliferation and increased sensitivity to IM treatment compared to control cells. Importantly, we further identified a new protein interaction between DNM2 and BCR-ABL in both BCR-ABL and BCR-ABL/AHI-1 co-transduced cells and this interaction is enhanced in BCR-ABL/AHI-1 co-transduced cells using co-IP/Western analysis. To the best of our knowledge, this is the first study to implicate this new AHI-1-DNM2-BCR-ABL complex in the deregulation of endocytosis signaling in CML, which may play an unusual role in regulation of the cellular properties of primitive CML cells, including their response/resistance to TKI, by aberrantly disrupting critical endocytosis processes in CML. Disclosures No relevant conflicts of interest to declare.