A Modular Design for the Clathrin- and Actin-Mediated Endocytosis Machinery

“The Rvs proteins arrive after the actin marker Abp1p has started to accumulate but reach their peak fluorescence intensity together with Abp1p (Figures 6B and 6C), consistent with the timing of scission during mammalian endocytosis (Merrifield et al., 2005).”

“Pathway modularity At the heart of clathrin-coated vesicle formation is a flexible network of interactions, which can be thought of as being made up of different module”

“…of the heterodimeric Bin/Amphiphysin/Rvs (BAR) domain-containing complex Rvs161/Rvs167 promotes scission by generating Developmental Cell 21, July 19, 2011 ª2011 Elsevier Inc. 83 a highly curved vesicle neck that is stressed by the pulling forces of actin polymerization (Kaksonen et al., 2005).”

“cerevisiae , the ARP2/3 complex and NPFs including Pan1, myosin-5, Las17 (the WASP homologue) and Abp1 are recruited to endocytic sites prior to, or concomitant with, actin polymerizatio”

“The discovery of the syndapin (also known as pacsin) family of proteins in mammals showed that N-WASP is involved in vesicular trafficking, particularly in endocytosi”

“Nonetheless, TIRFM imaging has been successfully accomplished even in yeast by squeezing cells between the coverslip and glass slide (13, 14).”

“Subsequently, using a live imaging screen of deletion mutants, where they dissected defects in coat dynamics and actin assembly, they provided evidence for the existence of distinct protein modules for coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin-or actin-mediate…”

“…that the Rvs161/167 proteins are recruited to late-stage endocytic actin patches, and that in yeast lacking these proteins there is a retraction or “yo-yo” movement of the endocytic membranes Cell generated at these sites after the initial internalization movement (Kaksonen et al., 2005).”

“The intensity profile as a function of time was used to characterize the dynamics of each of the AP-2 spots selected according to the following criteria: (1) fluorescent objects where diffraction limited; (2) dynamic, nonarrested objects appeared and disappeared within the time window of the time-series with lifetimes…”

“…in live cells (Toret and Drubin 2006), Drubin and colleagues have systematically identified actin patches, which have distinct protein composition and motility properties, and accumulate in the different genetic mutants as intermediates in the endocytic process (Kaksonen et al. 2003, 2005).”

“As in mammalian cells, endocytosis in yeast starts with the assembly of clathrin coat proteins, and many of the subsequent stages are orchestrated by proteins whose homologs are also present at mammalian clathrin-coated pits (Kaksonen et al. 2005; Ferguson et al. 2009; Taylor et al. 2011).”

“The dynamic organization of the endocytic machinery in budding yeast has been elucidated using live-cell imaging of fluorescent protein–labeled endocytic proteins (Kaksonen et al., 2003, 2005).”

“Recent studies have led to a much clearer picture of the molecular pathways underlying receptor-mediated endocytosis (263, 267, 268, 413), whereas the mechanisms underlying fluid-phase endocytosis remain unclear.”

“The endocytic proteins Sla1 and Bbc1, also recruited early in the endocytic process, cooperate to inhibit Arp2/3 activation by Las17 (20, 72, 73).”

“For example, yeast Rvs167/161 complex is intimately involved in actin assembly during endocytosis and mammalian tuba binds Arp2/3 activator N-WASP (Salazar et al., 2003; Kaksonen et al., 2005).”

“TIRF also provided the spatial and temporal resolution to study the dynamics of actin and actin-associated proteins near the plasma membrane in several endocytosis studies (Kaksonen et al., 2005; Merrifield et al., 2002).”

“cerevisiae , the involvement of clathrin is proposed to be limited to the initiation of endocytic-site assembly rather than membrane invagination or vesicle formatio”

“Consistent with this scenario, absence of the endophilin homologRvs167 in yeast leads toendocytic invaginations that bounce back and forth and often do not proceed to fission, suggesting a role of Rvs167 in stabilizing a preformed invagination (Kaksonen et al., 2005).”

“…required for efficient recycling of B2AR but not of TfR (Cao et al., 1999; Gage et al., 2005), and as it has been implicated in endosome motility (Stamnes, 2002; Girao et al., 2008) and vesicle scission at the cell surface (Yarar et al., 2005; Perrais and Merrifield, 2005; Kaksonen et al., 2005).”

“The receptor-mediated endocytosis is carried out by the sequential actions of distinct protein modules that regulate distinct processes such as coat formation, membrane invagination, and vesicle scission (Kaksonen et al. 2005).”

“Participation of the Arp2/3 complex and activators in cell division Assembly of actin patches in fungi during endocytosis ( Kaksonen et al., 2003, 2005) depends on the Arp2/3 complex to nucleate branched actin fi laments (Weaver et al., 2003).”

“The first evidence for an involvement of actin in clathrin-mediated endocytosis in higher eukaryotic cells dates back to the 1980s [57], but its critical function in clathrin-mediated endocytosis was only appreciated after the development of life cell imaging techniques using fluorescent actin in combination with tota…”

“Components of the mammalian endocytic actin machinery, including N-WASP, WIP, and syndapin, share homology to yeast endocytic proteins, and in both instances, these proteins play a role in clathrinmediated endocytosis (Kaksonen et al., 2005; Merrifield, 2004; Newpher et al., 2005).”

“In yeast, dynamin is not expressed and scission of tubular endocytic structures occurs following the recruitment of the actin module (Kaksonen et al., 2005).”

“In yeast, direct genomic tagging of pairs of genes with different fluorescent tags has allowed researchers to define a very regular series of spatiotemporal events for CME in living cell”

“The CLC has a punctate distribution within the plasma membrane of yeast and mammalian cells (Kaksonen et al., 2005; Merrifield et al., 2002).”

“Although compartmentation of the plasma membrane is a widespread phenomenon found in cells from bacteria to humans, physiological roles of this segregation are still debated ( Munro, 2003 ; Douglass and Vale, 2005 ; Kenworthy, 2008 ).”

“…in CCPs by dual imaging of clathrin and dynamin in cells coexpressing either db1 or ala. Consistent with previous studies (Ehrlich et al., 2004; Kaksonen et al., 2005; Merrifield et al., 2002), dynamin accumulated on CCPs and sharply peaked at the end of the ascending phase of clathrin…”

“As Sla1p internalizes, the Las17p inhibitor Bbc1p localizes to the patch [6, 37].”

“The rapid disappearance of CLC-GFP foci most likely corresponds to vesicle release from the plasma membrane (Merrifield et al., 2002, 2005; Kaksonen et al., 2005).”

“In the absence of Sac6p, actin polymerises at endocytic sites, but there is no internalisation (Kaksonen et al., 2005; Kübler and Riezman, 1993).”

“Clathrin was linked to endocytosis in animal cells in 1975 (Pearse 1975), but only in the last decade has a role for clathrin in yeast become fully accepted (Kaksonen et al. 2005; Newpher et al. 2005).”

“Live-cell imaging studies provided important details about the spatial and temporal organization of the factors involved in this sequence of events (Gaidarov et al., 1999; Ehrlich et al., 2004; Kaksonen et al., 2005; Taylor et al., 2011).”

“In contrast, Abp1p, Arp2/3p, and actin move slowly into the cell with the actin patch and remain associated as the actin patch undergoes a subsequent fast long-range movement (132, 139, 143, 144).”

“These actin-rich patches contain proteins involved in receptor-mediated endocytosis and appear to be defined and initiated by clathrin, but soon, proteins that regulate (Las17p and Arp2/3) actin take over the process and participate actively in vesicle formation and pinch off event (39).”

“Because actin patches are highly dynamic structures of changing protein composition, we selected several proteins that are recruited at the early (Ede1, Sla1), later (Crn1), and at final stage (Abp1) of an actin patch assembly cycle ( Kaksonen et al., 2003, 2005 ).”

“, respectively). Moreover, from a recently published set of approximately 60 known and putative endocytosis proteins, 29 were found to be connected with at least one SH3 domain in our interaction network”

“The internalization of a membrane coated by an extended, flat clathrin array, as occurs with coated plaques, involves an abrupt inwards movement into the cell of the entire coat [25, 31 ,32,34,61].”

“have revealed the spatial and temporal recruitment of coat and adaptor proteins and actin in yeas”

“It was previously shown that in bbc1D cells, the coat protein Sla1 moves in further than in wild-type cells and that more actin is assembled, resulting in a wider actin network (Kaksonen et al., 2005; Picco et al., 2018).”

“The role of Fimbrin in bundling actin filaments is thought to be involved in generating the force required to pull and pinch off the invaginated plasma membrane for the internalization process of endocytosis (Kaksonen et al., 2005).”

“Live imaging of coat and actin adaptor proteins within this set identified groups of phenotypes consistent with a modular design of carrier maturation [48].”

“However, during yeast endocytosis, actin patches are formed at the sites of internalization and have been seen around vesicles in EM studies (Gachet and Hyams, 2005; Kaksonen et al., 2005; Kanbe et al., 1989; Takagi et al., 2003).”

“Endocytosis is inhibited when cells are treated with actin-depolymerizing drugs, because internalization of endocytic vesicles requires actin patches (Ayscough, 2000, 2005; Huckaba et al., 2004; Kaksonen et al., 2005).”

“In clathrin-mediated endocytosis, the sequence of subunit recruitment (Kaksonen et al., 2005; Taylor et al., 2011) is also coupled to membrane deformation.”

“The formation of a finger-like invagination in relation to the endocytic event has been explained in molecular detail by fluorescence (Kaksonen et al., 2005) and electron microscopy (Idrissi et al., 2008) studies.”

“…actin-driven formation and internalization of endocytic vesicles and the appearance of actin “comet tails” on such vesicles is well-established and appears to be a highly complex process involving several protein systems (McPherson 2002; Kaksonen et al. 2005, 2006; Smythe and Ayscough 2006).”

“We fused a palmitoylation signal (the fi rst 28 amino acids of Psr1) to the N terminus of full-length GFP-Ypp1 ( Siniossoglou et al., 2000 ).”

“In view that Abp1 is a prototypic marker of endocytosis (Kaksonen et al., 2003; 2005; Huckaba et al., 2004; Sun et al., 2006), these and the above results strongly suggest that the marked polarization of cortical actin in A. nidulans reflects predominance of endocytic sites in the tip.”

“Time-lapse analyses showed that the lifetime of Syp1-GFP patches typically exceeded the two-minute span of the experiment, similar to the behaviour of Ede1-RFP and other components of the ‘early coat complex’, as defined earlier (Kaksonen et al, 2005).”

“…microscopy showed that fission yeast cells strictly control both timing and the numbers of molecules that appear and disappear over time at sites of endocytosis, starting with a hemisphere of clathrin and culminating in a wave of actin assembly (Kaksonen et al., 2003, 2005; Sirotkin et al., 2005).”

“In yeast, Las17 and verprolin 1 (the yeast homolog of WIP) are recruited to CCPs with the proteins Bzz1 and Rvs167, which are now known to be members of the EFC/ F-BAR and BAR domain-containing proteins [49,50].”

“…sjl2𝚫 cells are defective in functions of multiple endocytic modules Previously, based on the dynamic behavior of many different proteins during endocytic internalization, we proposed that the yeast endocytic machinery is composed of four protein modules (Kaksonen et al., 2005).”

“(B) Model of actin patch formation and movement, adapted from previous studies [”

“The online version of this article contains supplemental material.”

“Abp1pmRFP serves as a reporter for actin polymerization in the final stages of endocytic internalization and is an established reference for endocytic protein dynamics (Kaksonen et al., 2003, 2005).”

“The membrane-bound scaffolds formed by the endocytic BAR/F-BAR domain proteins are transient, and their assembly and disassembly during this process are precisely regulated (Henne et al., 2010; Kaksonen et al., 2005; Taylor et al., 2011).”