Spatial regulation of clathrin-mediated endocytosis through position-dependent site maturation

Pedersen, Ross T.A.; Hassinger, Julian; Marchando, Paul; Drubin, David G.

doi:10.1083/jcb.202002160

Cited by 21 publications

(16 citation statements)

References 60 publications

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“…Establishment of daughter cell identity in S. cerevisiae through exocytosis-mediated septin ring formation is likely to belong to the same class of morphogenetic processes ( 38 ). Membrane flows may also help couple endocytosis around sites of exocytosis, which also promote polarity establishment ( 39, 40 ). Other proteins localize to secretion sites despite membrane flows, thanks to fast membrane unbinding, directed cytoskeletal transport or preferred affinity to static components.…”

Section: Discussionmentioning

confidence: 99%

Cell patterning by secretion-induced plasma membrane flows

Gerganova

Lamas

Rutkowski

et al. 2020

Preprint

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Cells self-organize using reaction-diffusion and fluid-flow principles. Whether bulk membrane flows contribute to cell patterning has not been established. Here, using mathematical modelling, optogenetics and synthetic probes, we show that polarized exocytosis causes lateral membrane flows away from regions of membrane insertion. Plasma membrane-associated proteins with sufficiently low diffusion and/or detachment rates couple to the flows and deplete from areas of exocytosis. In rod-shaped fission yeast cells, zones of Cdc42 GTPase activity driving polarized exocytosis are limited by GTPase activating proteins (GAPs). We show that membrane flows pattern the GAP Rga4 distribution and coupling of a synthetic GAP to membrane flows is sufficient to establish the rod shape. Thus, membrane flows induced by Cdc42-dependent exocytosis form a negative feedback restricting the zone of Cdc42 activity.One Sentence SummaryExocytosis causes bulk membrane flows that drag associated proteins and form a negative feedback restricting the exocytic site.

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Section: Discussionmentioning

confidence: 99%

Cell patterning by secretion-induced plasma membrane flows

Gerganova

Lamas

Rutkowski

et al. 2020

Preprint

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“…That this cargo for the central AP-2 adaptor is in surplus suggests another mechanism for AP-2 to more stably nucleate clathrin coated pits on the membrane via its more abundant cargo. Recent experiments indicate that cargo selection is most impactful on coated pit maturation rather than initiation 69 , and establishing the timescales in future work by combining the network and abundances with spatial modeling 42 would further clarify how interactions with cargo can checkpoint the assembly of clathrin-coated vesicles 86 .…”

Section: Resultsmentioning

confidence: 95%

“…The lipids are required for the actual localization of cytoplasmic proteins to the membrane surface, where PI(4,5)P 2 is essential 68 . Alternative to our functional classification, CME proteins can be classified based on their arrival time at sites of CME 10,11,69 .…”

Section: Resultsmentioning

confidence: 99%

Integrating protein copy numbers with interaction networks to quantify stoichiometry in mammalian endocytosis

Duan

et al. 2020

Preprint

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Proteins that drive processes like clathrin-mediated endocytosis (CME) are expressed at various copy numbers within a cell, from hundreds (e.g. auxilin) to millions (e.g. clathrin). Between cell types with identical genomes, copy numbers further vary significantly both in absolute and relative abundance. These variations contain essential information about each protein's function, but how significant are these variations and how can they be quantified to infer useful functional behavior? Here, we address this by quantifying the stoichiometry of proteins involved in the CME network. We find robust trends across three cell types in proteins that are sub- vs super-stoichiometric in terms of protein function, network topology (e.g. hubs), and abundance. To perform this analysis, we first constructed the interface resolved network of 82 proteins involved in CME in mammals, plus lipid and cargo binding partners, totaling over 600 specific binding interactions. Our model solves for stoichiometric balance by optimizing each copy of a protein interface to match up to its partner interfaces, keeping the optimized copies as close as possible to observed copies. We find highly expressed, structure-forming proteins such as actin and clathrin do tend to be super-stoichiometric, or in excess of their partners, but they are not the most extreme cases. We test sensitivity of network stoichiometry to protein removal and find that hub proteins tend to be less sensitive to removal of any single partner, thus acting as buffers that compensate dosage changes. As expected, tightly coupled protein pairs (e.g. CAPZA2 and CAPZB) are strongly correlated. Unexpectedly, removal of functionally similar cargo adaptor proteins produces widely variable levels of disruption to the network stoichiometry. Our results predict that knockdown of the adaptor protein DAB2 will globally impact the stoichiometry of most other cargo adaptor proteins in Hela cells, with significantly less impact in fibroblast cells. This inexpensive analysis can be applied to any protein network, synthesizing disparate sources of biological data into a relatively simple and intuitive model of binding stoichiometry that can aid in dynamical modeling and experimental design.

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“…This idea is supported by experiments that show general defects in endocytosis resulting from deleting Ubdomains in combination with loss of other genes as well as the observation that the endocytic machinery also can undergo ubiquitination itself (Dores et al, 2010;Maldonado-Baez et al, 2008;Polo et al, 2002;Savio et al, 2016;Sugiyama et al, 2005;Tanner et al, 2019;Weinberg and Drubin, 2014;Woelk et al, 2006). In some ways, these ideas may be a false dichotomy in view of studies that show cargo itself can influence the assembly process, providing a way that recognition of Ub as an internalization signal is the mode through which Ub-binding domains control assembly (Carroll et al, 2012;Henry et al, 2012;Loerke et al, 2009;Maib et al, 2018;Pedersen et al, 2020;Puthenveedu and von Zastrow, 2006). The presence of Ub-cargo might help form endocytic vesicles of different constituents that can determine different fates (Sigismund et al, 2021), or drive more processive assembly of clathrin-coated vesicles by satisfying cargo checkpoints that Ub-binding components control (Chen and Schmid, 2020).…”

Section: Discussionmentioning

confidence: 94%

ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals

Pashkova

Gakhar

et al. 2021

Preprint

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Ubiquitin (Ub) serves as a signal for clathrin-mediated endocytosis (CME) by engaging Ub-binding proteins with the internalization apparatus. Ub is a versatile internalization signal because it can be added to a wide variety of membrane proteins, expanding the capacity of cells to use a variety of regulatory mechanisms to specify the conditions under which a particular protein will be internalized. Several candidate adaptors that can recognize ubiquitinated membrane proteins have been identified that work in endocytic processes that are both clathrin-dependent and independent. These include Epsin and Eps15, which bind and help sort Ub-cargo into internalization sites. Here we identify additional components of the endocytosis apparatus that bind Ub. The N-terminal ANTH domains found in CALM, AP180, HIP1R and yeast Sla2 all bind monoubiquitin with micromolar affinity. ANTH domains belong to a larger superfamily of domains including ENTH and VHS domains, many of which have Ub-binding regions outside of their VHS/ENTH/ANTH domains that enable them to mediate Ub-dependent sorting events throughout the cell. Solution NMR studies combined with a crystal structure of the CALM ANTH domain in a complex with Ub show that Ub binds to a C-terminal region of the ANTH domain that is not present in ENTH domains. Combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the internalization apparatus of yeast caused defects in the Ub-dependent internalization of the GPCR Ste2 but had no effect on internalization of Ste2 via other internalization signals. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.

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Spatial regulation of clathrin-mediated endocytosis through position-dependent site maturation

Cited by 21 publications

References 60 publications

Cell patterning by secretion-induced plasma membrane flows

Cell patterning by secretion-induced plasma membrane flows

Integrating protein copy numbers with interaction networks to quantify stoichiometry in mammalian endocytosis

ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals

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