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Significance Clathrin-mediated endocytosis (CME), the major pathway for uptake into cells, is a multistep process involving a myriad of endocytic accessory proteins (EAPs). Although the biochemical activities of many EAPs have been extensively studied, their stage-specific role(s) during clathrin-coated pit (CCP) initiation, stabilization, and/or maturation are poorly defined. Here, using quantitative total internal reflection fluorescence microscopy and a rigorous experimental and multiparametric analytical pipeline, we study the effect of CRISPR interference (CRISPRi)- or small interfering RNA (siRNA)-mediated knockdown of 67 individual EAPs on CCP dynamics. Our comprehensive analyses combined with unsupervised phenotypic clustering reveal the complex and overlapping roles of EAPs during early, critical stages of CME, providing a valuable resource to spur further research into their function., Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK’s multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME.

Clathrin mediated endocytosis (CME) has been extensively studied in living cells by quantitative total internal reflection fluorescence microscopy (TIRFM). Fluorescent protein fusions to subunits of the major coat proteins, clathrin light chains or the heterotetrameric adaptor protein (AP2) complexes, have been used as fiduciary markers of clathrin coated pits (CCPs). However, the functionality of these fusion proteins has not been rigorously compared. Here, we generated stable cells lines overexpressing mRuby‐CLCa and/or μ2‐eGFP, σ2‐eGFP, two markers currently in use, or a novel marker generated by inserting eGFP into the unstructured hinge region of the α subunit (α‐eGFP). Using biochemical and TIRFM‐based assays, we compared the functionality of the AP2 markers. All of the eGFP‐tagged subunits were efficiently incorporated into AP2 and displayed greater accuracy in image‐based CCP analyses than mRuby‐CLCa. However, overexpression of either μ2‐eGFP or σ2‐eGFP impaired transferrin receptor uptake. In addition, μ2‐eGFP reduced the rates of CCP initiation and σ2‐eGFP perturbed AP2 incorporation into CCPs and CCP maturation. In contrast, CME and CCP dynamics were unperturbed in cells overexpressing α‐eGFP. Moreover, α‐eGFP was a more sensitive and accurate marker of CCP dynamics than mRuby‐CLCa. Thus, our work establishes α‐eGFP as a robust, fully functional marker for CME., Fluorescent protein fusions are used as fiduciary markers to study clathrin‐mediated endocytosis (CME). However, their functionality has not been rigorously compared. Here, we introduce a novel marker generated by inserting eGFP into the hinge region of the AP2 α subunit (α‐eGFP). Using biochemical and imaging‐based studies its functionality was compared to that of commonly used μ2‐eGFP and σ2‐eGFP fusions. Only cells overexpressing α‐eGFP exhibited unperturbed CME and clathrin coated pit dynamics, establishing it as a robust fiduciary marker for CME.

Chen et al. define the role of the N-terminal domain (TD) of clathrin heavy chain in early and late stages of clathrin-mediated endocytosis (CME) and design a membrane-permeant peptide, Wbox2, that acutely and potently inhibits CME., Clathrin-mediated endocytosis (CME) occurs via the formation of clathrin-coated vesicles from clathrin-coated pits (CCPs). Clathrin is recruited to CCPs through interactions between the AP2 complex and its N-terminal domain, which in turn recruits endocytic accessory proteins. Inhibitors of CME that interfere with clathrin function have been described, but their specificity and mechanisms of action are unclear. Here we show that overexpression of the N-terminal domain with (TDD) or without (TD) the distal leg inhibits CME and CCP dynamics by perturbing clathrin interactions with AP2 and SNX9. TDD overexpression does not affect clathrin-independent endocytosis or, surprisingly, AP1-dependent lysosomal trafficking from the Golgi. We designed small membrane–permeant peptides that encode key functional residues within the four known binding sites on the TD. One peptide, Wbox2, encoding residues along the W-box motif binding surface, binds to SNX9 and AP2 and potently and acutely inhibits CME.

Clathrin-mediated endocytosis (CME) occurs via the formation of clathrin-coated vesicles from clathrin-coated pits (CCPs). Clathrin is recruited to CCPs through interactions between the AP2 complex and its N-terminal domain (TD), which in turn recruits endocytic accessory proteins. Inhibitors of CME that interfere with clathrin function have been described, but their specificity and mechanisms of action are unclear. Here we show that overexpression of the TD with or without the distal leg specifically inhibits CME and CCP dynamics by perturbing clathrin interactions with AP2 and SNX9. We designed small membrane-penetrating peptides that mimic the four known binding sites on the TD. A peptide, Wbox2, designed to mimic to the W-box motif binding surface on TD binds to SNX9 and AP2, and potently and acutely inhibits CME, while not perturbing AP1-dependent lysosomal trafficking from the Golgi or bulk, fluid phase endocytosis.SummaryChen et al define the role the N-terminal domain (TD) of clathrin heavy chain in early and late stages of clathrin-mediated endocytosis, and guided by its structure, design a membrane-penetrating peptide, Wbox2, that acutely and potently inhibits CME.

Background: Activating mutations in ERBB2 (HER2) are found in ~2% of solid tumors, including ~5% of metastatic breast cancers. The spectrum of HER2 mutations varies according to tumor type, with kinase domain (KD) missense mutations being most common in breast cancer, while exon 20 insertions are most common in non-small cell lung cancer (NSCLC). Breast cancer cells engineered with activating HER2 mutations show elevated HER2 phosphorylation and enhanced oncogenic growth. Activating mutations of other receptor tyrosine kinases such as EGFR affect receptor internalization, but whether HER2 mutations alter receptor internalization is not yet known. In addition, clinical responses to HER2 antibody-drug conjugates (ADCs) like T-DM1 have been noted in HER2-mutant NSCLC, raising the possibility that HER2 mutations alter receptor internalization, thus enhancing ADC sensitivity. Therefore, we aimed to 1) determine whether mutant HER2 receptor internalization is altered, and 2) determine whether altered internalization contributes to HER2 ADC sensitivity in HER2-mutant breast cancer. Methods: HER2 subcellular localization was investigated by immunofluorescence (IF) in MCF7 cells where HER2L755S, HER2V777L, or HER2L869R (or HER2WT) have been ‘knocked-in' using AAV-mediated recombination. We used ELISA-based endocytosis assays to quantify the internalization efficiency of trastuzumab-labeled HER2 in cell lines with WT and mutant HER2. Cell viability upon treatment with T-DM1 was tested using conventional 2D and 3D growth assays. Results: Partial colocalization of HER2 with clathrin, measured by IF, was observed in MCF7 cells, which was not affected by HER2 mutations. In the endocytosis assay, trastuzumab was inefficiently internalized over a period of 4 hours. In MCF7 or MCF10A breast epithelial cells, introduction of HER2L755S, HER2V777L, or HER2L869R did not significantly affect internalization following trastuzumab labeling, or after stimulation with EGF or neuregulin. In line with these results, MCF7 cells expressing HER2L755S, HER2V777L, or HER2L869R were not sensitive to T-DM1 in 2D cell viability assays or 3D Matrigel assays. In contrast, HER2 internalization was significantly more efficient in BT474 cells with wild-type HER2 amplification, which was accompanied by exquisite sensitivity of BT474 cells to T-DM1 both in vitro and in vivo. HER2 endocytic efficiency was significantly increased in MCF10A cells ectopically expressing HER2S310F, a mutation in the extracellular domain, or HER2A775>YVMA, a common insertion mutation in NSCLC. Conclusion: Breast cancer cells expressing HER2 KD missense mutations do not exhibit enhanced HER2 internalization and are not sensitive to T-DM1. However, cells with the HER2S310F mutation and the HER2A775>YVMA insertion showed enhanced HER2 endocytosis, consistent with clinical responses to T-DM1 in NSCLC patients with these mutations. Citation Format: Hiroaki Akamatsu, Rosa Mino, Marcel Mettlen, Dan Ye, Dhivya R. Sudhan, Albert Lin, Arnaldo Marin, Alberto Servetto, Kyung-min Lee, Sumanta Chatterjee, Sandra L. Schmid, Carlos L. Arteaga, Ariella B. Hanker. HER2 missense mutations in breast cancer cells do not alter HER2 internalization or sensitivity to T-DM1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3675.

Neuron-glial interactions are crucial for growth, guidance and ensheathment of axons across species. In the Drosophila CNS midline, neuron-glial interactions underlie ensheathment of commissural axons by midline glial (MG) cells in a manner similar to mammalian oligodendrocytes. Although there has been some advance in the study of neuron-glial interactions and ensheathment of axons in the CNS midline, key aspects of axonal ensheathment are still not fully understood. One of the limitations has been the unavailability of MG membrane markers that could highlight the glial processes wrapping the axons. Previous studies have identified two key molecular players from the neuronal and glial cell types in the CNS midline. These are the neuronal transmembrane protein Neurexin IV (Nrx IV) and the membrane-anchored MG protein Wrapper, both of which interact in trans to mediate neuron-glial interactions and ensheathment of commissural axons. In the current study, we attempt to further our understanding of MG biology and try to overcome some of the technical difficulties posed by the lack of a robust MG driver that will specifically allow expression or knockdown of genes in MG. We report the generation of BAC transgenic flies of wrapper-GAL4 and demonstrate how these flies could be used as a genetic tool to understand MG biology. We have utilized the GAL4/UAS system to drive GFP-reporter lines (membrane-bound mCD8-GFP; microtubule-associated tau-GFP) and nuclear lacZ using wrapper-GAL4 to highlight the MG cells and/or their processes that surround and perform axonal ensheathment functions in the embryonic midline. We also describe the utility of the wrapper-GAL4 driver line to down-regulate known MG genes specifically in Wrapper-positive cells. Finally, we validate the functionality of the wrapper-GAL4 driver by rescue of wrapper mutant phenotypes and lethality. Together, these studies provide us with a versatile genetic tool to investigate MG functions and will aid in future investigations where genetic screens using wrapper-GAL4 could be designed to identify novel molecular players at the Drosophila midline and unravel key aspects of MG biology.

Axonal growth and targeting are fundamental to the organization of the nervous system, and require active engagement of the cytoskeleton. Polymerization and stabilization of axonal microtubules is central to axonal growth and maturation of neuronal connectivity. S tudies have suggested that members of the tubulin polymerization promoting protein (TPPP, also known as P25α) family are involved in cellular process extension. However, no in vivo knockout data exists regarding its role in axonal growth during development. Here, we report the characterization of Ringmaker (Ringer; CG45057), the only Drosophila homolog of long p25α proteins. Immunohistochemical analyses indicate that Ringer expression is dynamically regulated in the embryonic central nervous system (CNS). ringer -null mutants show cell misplacement, and errors in axonal extension and targeting. Ultrastructural examination of ringer mutants revealed defective microtubule morphology and organization. Primary neuronal cultures of ringer mutants exhibit defective axonal extension, and Ringer expression in cells induced microtubule stabilization and bundling into rings. In vitro assays showed that Ringer directly affects tubulin, and promotes microtubule bundling and polymerization. Together, our studies uncover an essential function of Ringer in axonal extension and targeting through proper microtubule organization.

depends on activation of the tyrosine kinase receptor, Sevenless (Sev), in the R7. Prior to Sev signaling, Pros is equally expressed in all five R7 equivalence cells. Sev activation increases Pros expression specifically in the presumptive R7, suggesting that differences in Pros levels affect cell fate specification. Here, we tested whether changing Pros levels in the R7 equivalence group controls R7 vs. CC development. Indeed, genetic increase and/or decrease of pros leads to defects in the adult lens. Moreover, we found genetic interactors for Pros that affect lens formation, further supporting a dosedependent requirement for Pros during differentiation. Ongoing studies are aimed at examine how Pros controls the development and/or maintenance of the fly lens using a variety of molecular and morphological criteria. Interestingly, the vertebrate homologue of Pros, Prox1, has been shown to be important during lens development. Moreover, removal of one copy of mouse prox1 causes cataracts. Together, these data suggest that fly and vertebrate lens formation relies on evolutionarily conserved developmental pathways. Thus, we propose to use the fly eye as a new genetic system for uncovering fundamental processes necessary for vertebrate lens formation and maintenance. Support from Research Preventing Blindness and Ziegler Fdn for the Blind.