scholarly journals Evolving models for assembling and shaping clathrin-coated pits

2020 ◽  
Vol 219 (9) ◽  
Author(s):  
Zhiming Chen ◽  
Sandra L. Schmid
Keyword(s):  
Protein Interactions ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Adaptor Protein ◽  
Structural Studies ◽  
Coated Pits ◽  
Molecular Events ◽  
Em Tomography ◽  
Evolving Models ◽  
Flexible Model

Clathrin-mediated endocytosis occurs via the assembly of clathrin-coated pits (CCPs) that invaginate and pinch off to form clathrin-coated vesicles (CCVs). It is well known that adaptor protein 2 (AP2) complexes trigger clathrin assembly on the plasma membrane, and biochemical and structural studies have revealed the nature of these interactions. Numerous endocytic accessory proteins collaborate with clathrin and AP2 to drive CCV formation. However, many questions remain as to the molecular events involved in CCP initiation, stabilization, and curvature generation. Indeed, a plethora of recent evidence derived from cell perturbation, correlative light and EM tomography, live-cell imaging, modeling, and high-resolution structural analyses has revealed more complexity and promiscuity in the protein interactions driving CCP maturation than anticipated. After briefly reviewing the evidence supporting prevailing models, we integrate these new lines of evidence to develop a more dynamic and flexible model for how redundant, dynamic, and competing protein interactions can drive endocytic CCV formation and suggest new approaches to test emerging models.

scholarly journals LRRK2 mediates tubulation and vesicle sorting from membrane damaged lysosomes

2020 ◽  
Author(s):  
Luis Bonet-Ponce ◽  
Alexandra Beilina ◽  
Chad D. Williamson ◽  
Eric Lindberg ◽  
Jillian H. Kluss ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Leucine Rich Repeat ◽  
Biological Functions ◽  
New Process ◽  
Sporadic Parkinson’S Disease

ABSTRACTMutations in the leucine rich repeat kinase 2 (LRRK2) gene are a cause of familial and sporadic Parkinson’s disease (PD). Nonetheless, the biological functions of LRRK2 remain incompletely understood. Here, we observed that LRRK2 is recruited to lysosomes that have a ruptured membrane. Using unbiased proteomics, we observed that LRRK2 is able to recruit the motor adaptor protein JIP4 to permeabilized lysosomes in a kinase-dependent manner through the phosphorylation of RAB35 and RAB10. Super-resolution live cell imaging microscopy and FIB-SEM revealed that once at the lysosomal membrane, JIP4 promotes the formation of LAMP1-negative lysosomal tubules that release membranous content from ruptured lysosomes. Released vesicular structures are able to interact with other lysosomes. Thus, we described a new process that uses lysosomal tubulation to release vesicular structures from permeabilized lysosomes. LRRK2 orchestrates this process that we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2) that, given the central role of the lysosome in PD, is likely to be disease relevant.

scholarly journals Detection and Characterization of Protein Interactions In Vivo by a Simple Live-Cell Imaging Method

PLoS ONE ◽  
2013 ◽  
Vol 8 (5) ◽  
pp. e62195 ◽  
Author(s):  
Oriol Gallego ◽  
Tanja Specht ◽  
Thorsten Brach ◽  
Arun Kumar ◽  
Anne-Claude Gavin ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Imaging Method

scholarly journals DASC, a sensitive classifier for measuring discrete early stages in clathrin-mediated endocytosis

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Xinxin Wang ◽  
Zhiming Chen ◽  
Marcel Mettlen ◽  
Jungsik Noh ◽  
Sandra L Schmid ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Single Channel ◽  
Accessory Proteins ◽  
Sensitive Detector ◽  
Coated Pits ◽  
Misleading Information ◽  
Bona Fide ◽  
Biochemical Measurements

Clathrin-mediated endocytosis (CME) in mammalian cells is driven by resilient machinery that includes >70 endocytic accessory proteins (EAP). Accordingly, perturbation of individual EAPs often results in minor effects on biochemical measurements of CME, thus providing inconclusive/misleading information regarding EAP function. Live-cell imaging can detect earlier roles of EAPs preceding cargo internalization; however, this approach has been limited because unambiguously distinguishing abortive coats (ACs) from bona fide clathrin-coated pits (CCPs) is required but unaccomplished. Here, we develop a thermodynamics-inspired method, “disassembly asymmetry score classification (DASC)”, that resolves ACs from CCPs based on single channel fluorescent movies. After extensive verification, we use DASC-resolved ACs and CCPs to quantify CME progression in 11 EAP knockdown conditions. We show that DASC is a sensitive detector of phenotypic variation in CCP dynamics that is uncorrelated to the variation in biochemical measurements of CME. Thus, DASC is an essential tool for uncovering EAP function.

scholarly journals SUMOylation of the Kv4.2 Ternary Complex Increases Surface Expression and Current Amplitude by Reducing Internalization in HEK 293 Cells

2021 ◽  
Vol 14 ◽  
Author(s):  
Meghyn A. Welch ◽  
Leslie-Anne R. Jansen ◽  
Deborah J. Baro
Keyword(s):  
Protein Interactions ◽  
Adaptor Protein ◽  
Surface Expression ◽  
Experimental Manipulation ◽  
Coated Pits ◽  
Maximal Conductance ◽  
Dynamic Regulation ◽  
Hek 293 ◽  
Hek Cells ◽  
Α Subunits

Kv4 α-subunits exist as ternary complexes (TC) with potassium channel interacting proteins (KChIP) and dipeptidyl peptidase-like proteins (DPLP); multiple ancillary proteins also interact with the α-subunits throughout the channel’s lifetime. Dynamic regulation of Kv4.2 protein interactions adapts the transient potassium current, IA, mediated by Kv4 α-subunits. Small ubiquitin-like modifier (SUMO) is an 11 kD peptide post-translationally added to lysine (K) residues to regulate protein–protein interactions. We previously demonstrated that when expressed in human embryonic kidney (HEK) cells, Kv4.2 can be SUMOylated at two K residues, K437 and K579. SUMOylation at K437 increased surface expression of electrically silent channels while SUMOylation at K579 reduced IA maximal conductance (Gmax) without altering surface expression. KChIP and DPLP subunits are known to modify the pattern of Kv4.2 post-translational decorations and/or their effects. In this study, co-expressing Kv4.2 with KChIP2a and DPP10c altered the effects of enhanced Kv4.2 SUMOylation. First, the effect of enhanced SUMOylation was the same for a TC containing either the wild-type Kv4.2 or the mutant K437R Kv4.2, suggesting that either the experimental manipulation no longer enhanced K437 SUMOylation or K437 SUMOylation no longer influenced Kv4.2 surface expression. Second, instead of decreasing IA Gmax, enhanced SUMOylation at K579 now produced a significant ∼37–70% increase in IA maximum conductance (Gmax) and a significant ∼30–50% increase in Kv4.2g surface expression that was accompanied by a 65% reduction in TC internalization. Blocking clathrin-mediated endocytosis (CME) in HEK cells expressing the Kv4.2 TC mimicked and occluded the effect of SUMO on IA Gmax; however, the amount of Kv4.2 associated with the major adaptor for constitutive CME, adaptor protein 2 (AP2), was not SUMO dependent. Thus, SUMOylation reduced Kv4.2 internalization by acting downstream of Kv4.2 recruitment into clathrin-coated pits. In sum, the two major findings of this study are: SUMOylation of Kv4.2 at K579 regulates TC internalization most likely by promoting channel recycling. Additionally, there is a reciprocity between Kv4.2 SUMOylation and the Kv4.2 interactome such that SUMOylation regulates the interactome and the interactome influences the pattern and effect of SUMOylation.

scholarly journals DASC, a sensitive classifier for measuring discrete early stages in clathrin-mediated endocytosis

2020 ◽  
Author(s):  
Xinxin Wang ◽  
Zhiming Chen ◽  
Marcel Mettlen ◽  
Jungsik Noh ◽  
Sandra L. Schmid ◽  
...  
Keyword(s):  
Phenotypic Variation ◽  
Mammalian Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Accessory Proteins ◽  
Sensitive Detector ◽  
Coated Pits ◽  
Misleading Information ◽  
Bona Fide ◽  
Biochemical Measurements

AbstractClathrin-mediated endocytosis (CME) in mammalian cells is driven by resilient machinery that includes >70 endocytic accessory proteins (EAP). Accordingly, perturbation of individual EAPs often results in minor effects on biochemical measurements of CME, thus providing inconclusive/misleading information regarding EAP function. Live-cell imaging can detect earlier roles of EAPs preceding cargo internalization; however, this approach has been limited because unambiguously distinguishing abortive-clathrin coats (ACs) from bona fide clathrin-coated pits (CCPs) is required but unaccomplished. Here, we develop a thermodynamics-inspired method, “disassembly asymmetry score classification (DASC)”, that unambiguously separates ACs from CCPs without an additional marker. After extensive verification, we use DASC-resolved ACs and CCPs to quantify CME progression in 11 EAP knockdown conditions. We show that DASC is a sensitive detector of phenotypic variation in CCP dynamics that is orthogonal to the variation in biochemical measurements of CME. Thus, DASC is an essential tool for uncovering the function of individual EAPs.

scholarly journals Subcellular Journey of Rare Cold Inducible 2 Protein in Plant Under Stressful Condition

2021 ◽  
Vol 11 ◽  
Author(s):  
Hyun-Sung Kim ◽  
Won Park ◽  
Hyeon-Sook Lee ◽  
Jung-Ho Shin ◽  
Sung-Ju Ahn
Keyword(s):  
Membrane Proteins ◽  
Protein Interactions ◽  
Abiotic Stresses ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Abiotic Stress Tolerance ◽  
Protein Structures ◽  
Hydrophobic Membrane ◽  
Small Hydrophobic

Rare cold inducible 2 (RCI2) proteins are small hydrophobic membrane proteins in plants, and it has been widely reported that RCI2 expressions are dramatically induced by salt, cold, and drought stresses in many species. The RCI2 proteins have been shown to regulate plasma membrane (PM) potential and enhance abiotic stress tolerance when over-expressed in plants. RCI2 protein structures contain two transmembrane domains that are thought to be PM intrinsic proteins and have been observed at the PM and endomembranes. However, cellular trafficking of RCI2s are not fully understood. In this review, we discussed (i) general properties of RCI2s characterized in many species, (ii) the uses of RCI2s as a tracer in live cell imaging analyses and when they are fused to fluorescence proteins during investigations into vesicle trafficking, and (iii) RCI2 functionalities such as their involvement in rapid diffusion, endocytosis, and protein interactions. Consequently, the connection between physiological characteristics of RCI2s and traffic of RCI2s interacting membrane proteins might be helpful to understand role of RCI2s contributing abiotic stresses tolerance.

Focusing on clathrin-mediated endocytosis

2008 ◽  
Vol 412 (3) ◽  
pp. 415-423 ◽  
Author(s):  
Joshua Z. Rappoport
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
New Technologies ◽  
Adaptor Protein ◽  
Live Cell ◽  
Present Review ◽  
Live Cells ◽  
Experimental Systems ◽  
Exciting Time ◽  
Dramatic Shift

Investigations into the mechanisms which regulate entry of integral membrane proteins, and associated ligands, into the cell through vesicular carriers (endocytosis) have greatly benefited from the application of live-cell imaging. Several excellent recent reviews have detailed specific aspects of endocytosis, such as entry of particular cargo, or the different routes of internalization. The aim of the present review is to highlight how advances in live-cell fluorescence microscopy have affected the study of clathrin-mediated endocytosis. The last decade has seen a tremendous increase in the development and dissemination of methods for imaging endocytosis in live cells, and this has been followed by a dramatic shift in the way this critical cellular pathway is studied and understood. The present review begins with a description of the technical advances which have permitted new types of experiment to be performed, as well as potential pitfalls of these new technologies. Subsequently, advances in the understanding of three key endocytic proteins will be addressed: clathrin, dynamin and AP-2 (adaptor protein 2). Although great strides have clearly been made in these areas in recent years, as is often the case, each answer has bred numerous questions. Furthermore, several examples are highlighted where, because of seemingly minor differences in experimental systems, what appear at first to be very similar studies have, at times, yielded vastly differing results and conclusions. Thus this is an exceedingly exciting time to study endocytosis, and this area serves as a clear demonstration of the power of applying live-cell imaging to answer fundamental biological questions.

scholarly journals Adaptor protein Bbc1 regulates localization of Wsp1 and Vrp1 during endocytic actin patch assembly

2018 ◽  
Author(s):  
Cameron MacQuarrie ◽  
MariaSanta Mangione ◽  
Robert Carroll ◽  
Michael James ◽  
Kathleen L. Gould ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Schizosaccharomyces Pombe ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Adaptor Protein ◽  
Live Cell ◽  
Endocytic Vesicle ◽  
Actin Networks ◽  
Binding Partner ◽  
Actin Patch

ABSTRACTArp2/3 complex-nucleated branched actin networks provide the force necessary for endocytosis. The Arp2/3 complex is activated by Nucleation Promoting Factors (NPFs) including the Schizosaccharomyces pombe proteins WASp Wsp1 and myosin-1 Myo1. There are >40 known yeast endocytic proteins with distinct spatial and temporal localizations and functions; however, it is still unclear how these proteins work together to drive endocytosis. We used quantitative live cell imaging to determine the function of the uncharacterized S. pombe protein Bbc1. We discovered Myo1 interacts with and recruits Bbc1 to sites of endocytosis. Bbc1 competes with verprolin Vrp1 for Myo1 binding, thus releasing Vrp1 and its binding partner Wsp1 from Myo1. Normally Myo1 remains at the base of the endocytic invagination and Vrp1-Wsp1 internalize with the endocytic vesicle; however, in the absence of Bbc1, a portion of Vrp1-Wsp1 remains with Myo1 at the base of the invagination and endocytic invaginations are twice as long. We propose that Bbc1 disrupts a transient Myo1-Vrp1-Wsp1 interaction and limits Arp2/3 complex-nucleation of actin branches at the plasma membrane.

scholarly journals Visualization of Calcium Ion Loss from Rotavirus during Cell Entry

2018 ◽  
Vol 92 (24) ◽  
Author(s):  
Eric N. Salgado ◽  
Brian Garcia Rodriguez ◽  
Nagarjun Narayanaswamy ◽  
Yamuna Krishnan ◽  
Stephen C. Harrison
Keyword(s):  
Conformational Changes ◽  
Outer Layer ◽  
Live Cell Imaging ◽  
Molecular Mechanisms ◽  
Cell Imaging ◽  
Tight Binding ◽  
Live Cell ◽  
Membrane Disruption ◽  
Molecular Events

ABSTRACTBound calcium ions stabilize many nonenveloped virions. Loss of Ca2+from these particles appears to be a regulated part of entry or uncoating. The outer layer of an infectious rotavirus triple-layered particle (TLP) comprises a membrane-interacting protein (VP4) anchored by a Ca2+-stabilized protein (VP7). Membrane-coupled conformational changes in VP4 (cleaved to VP8* and VP5*) and dissociation of VP4 and VP7 accompany penetration of the double-layered inner capsid particle (DLP) into the cytosol. Removal of Ca2+in vitrostrips away both outer layer proteins; we and others have postulated that the loss of Ca2+triggers molecular events in viral penetration. We have now investigated, with the aid of a fluorescent Ca2+sensor, the timing of Ca2+loss from entering virions with respect to the dissociation of VP4 and VP7. In live-cell imaging experiments, distinct fluorescent markers on the DLP and on VP7 report on outer layer dissociation and DLP release. The Ca2+sensor, placed on VP5*, monitors the Ca2+concentration within the membrane-bound vesicle enclosing the entering particle. Slow (1-min duration) loss of Ca2+precedes the onset of VP7 dissociation by about 2 min and DLP release by about 7 min. Coupled with our previous results showing that VP7 loss follows tight binding to the cell surface by about 5 min, these data indicate that Ca2+loss begins as soon as the particle has become fully engulfed within the uptake vesicle. We discuss the implications of these findings for the molecular mechanism of membrane disruption during viral entry.IMPORTANCENonenveloped viruses penetrate into the cytosol of the cells that they infect by disrupting the membrane of an intracellular compartment. The molecular mechanisms of membrane disruption remain largely undefined. Functional reconstitution of infectious rotavirus particles (TLPs) from RNA-containing core particles (DLPs) and the outer layer proteins that deliver them into a cell makes these important pediatric pathogens particularly good models for studying nonenveloped virus entry. We report here how the use of a fluorescent Ca2+sensor, covalently linked to one of the viral proteins, allows us to establish, using live-cell imaging, the timing of Ca2+loss from an entering particle and other molecular events in the entry pathway. Specific Ca2+binding stabilizes many other viruses of eukaryotes, and Ca2+loss appears to be a trigger for steps in penetration or uncoating. The experimental design that we describe may be useful for studying entry of other viral pathogens.

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