A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

  1. Perunthottathu K Umasankar
  2. Li Ma
  3. James R Thieman
  4. Anupma Jha
  5. Balraj Doray
  6. Simon C Watkins
  7. Linton M Traub  Is a corresponding author
  1. University of Pittsburgh School of Medicine, United States
  2. Tsinghua University School of Medicine, China
  3. Olympus America, Inc., United States
  4. Washington University School of Medicine, United States

Abstract

Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization.

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Author details

  1. Perunthottathu K Umasankar

    University of Pittsburgh School of Medicine, Pittsburgh, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Li Ma

    Tsinghua University School of Medicine, Beijing, China
    Competing interests
    The authors declare that no competing interests exist.
  3. James R Thieman

    Olympus America, Inc., Center Valley, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Anupma Jha

    University of Pittsburgh School of Medicine, Pittsburgh, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Balraj Doray

    Washington University School of Medicine, St. Louis, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Simon C Watkins

    University of Pittsburgh School of Medicine, Pittsburgh, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Linton M Traub

    University of Pittsburgh School of Medicine, Pittsburgh, United States
    For correspondence
    traub@pitt.edu
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Suzanne R Pfeffer, Stanford University, United States

Publication history

  1. Received: July 23, 2014
  2. Accepted: October 8, 2014
  3. Accepted Manuscript published: October 10, 2014 (version 1)
  4. Version of Record published: November 3, 2014 (version 2)

Copyright

© 2014, Umasankar et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Perunthottathu K Umasankar
  2. Li Ma
  3. James R Thieman
  4. Anupma Jha
  5. Balraj Doray
  6. Simon C Watkins
  7. Linton M Traub
(2014)
A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing
eLife 3:e04137.
https://doi.org/10.7554/eLife.04137

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Linton M Traub
    Tools and Resources Updated

    Besides AP-2 and clathrin triskelia, clathrin coat inception depends on a group of early-arriving proteins including Fcho1/2 and Eps15/R. Using genome-edited cells, we described the role of the unstructured Fcho linker in stable AP-2 membrane deposition. Here, expanding this strategy in combination with a new set of llama nanobodies against EPS15 shows an FCHO1/2–EPS15/R partnership plays a decisive role in coat initiation. A nanobody containing an Asn-Pro-Phe peptide within the complementarity-determining region 3 loop is a function-blocking pseudoligand for tandem EPS15/R EH domains. Yet, in living cells, EH domains gathered at clathrin-coated structures are poorly accessible, indicating residence by endogenous NPF-bearing partners. Forcibly sequestering cytosolic EPS15 in genome-edited cells with nanobodies tethered to early endosomes or mitochondria changes the subcellular location and availability of EPS15. This combined approach has strong effects on clathrin coat structure and function by dictating the stability of AP-2 assemblies at the plasma membrane.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Hidehiko Okuma, Jeffrey M Hord ... Kevin P Campbell
    Research Advance

    Dystroglycan (DG) requires extensive post-translational processing and O-glycosylation to function as a receptor for extracellular matrix (ECM) proteins containing laminin-G-like (LG) domains. Matriglycan is an elongated polysaccharide of alternating xylose (Xyl) and glucuronic acid (GlcA) that binds with high-affinity to ECM proteins with LG-domains and is uniquely synthesized on α-dystroglycan (α-DG) by like-acetylglucosaminyltransferase-1 (LARGE1). Defects in the post-translational processing or O-glycosylation of α-DG that result in a shorter form of matriglycan reduce the size of α-DG and decrease laminin binding, leading to various forms of muscular dystrophy. Previously, we demonstrated that Protein O-Mannose Kinase (POMK) is required for LARGE1 to generate full-length matriglycan on α-DG (~150-250 kDa) (Walimbe et al., 2020). Here, we show that LARGE1 can only synthesize a short, non-elongated form of matriglycan in mouse skeletal muscle that lacks the DG N-terminus (α-DGN), resulting in a ~100-125 kDa α-DG. This smaller form of α-DG binds laminin and maintains specific force but does not prevent muscle pathophysiology, including reduced force production after eccentric contractions or abnormalities in the neuromuscular junctions. Collectively, our study demonstrates that α-DGN, like POMK, is required for LARGE1 to extend matriglycan to its full mature length on α-DG and thus prevent muscle pathophysiology.