Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics.

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.

An internally eGFP-tagged α-adaptin is a fully functional and improved fiduciary marker for clathrin-coated pit dynamics.

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.

A versatile genetic tool to study midline glia function in the Drosophila CNS.

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.

Drosophila Ringmaker regulates microtubule stabilization and axonal extension during embryonic development.

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. Studies 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.

Wbox2: A clathrin terminal domain-derived peptide inhibitor of clathrin-mediated endocytosis.

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.

A Laminin G-EGF-Laminin G module in Neurexin IV is essential for the apico-lateral localization of Contactin and organization of septate junctions.

Septate junctions (SJs) display a unique ultrastructural morphology with ladder-like electron densities that are conserved through evolution. Genetic and molecular analyses have identified a highly conserved core complex of SJ proteins consisting of three cell adhesion molecules Neurexin IV, Contactin, and Neuroglian, which interact with the cytoskeletal FERM domain protein Coracle. How these individual proteins interact to form the septal arrays that create the paracellular barrier is poorly understood. Here, we show that point mutations that map to specific domains of neurexin IV lead to formation of fewer septae and disorganization of SJs. Consistent with these observations, our in vivo domain deletion analyses identified the first Laminin G-EGF-Laminin G module in the extracellular region of Neurexin IV as necessary for the localization of and association with Contactin. Neurexin IV protein that is devoid of its cytoplasmic region is able to create septae, but fails to form a full complement of SJs. These data provide the first in vivo evidence that specific domains in Neurexin IV are required for protein-protein interactions and organization of SJs. Given the molecular conservation of SJ proteins across species, our studies may provide insights into how vertebrate axo-glial SJs are organized in myelinated axons.