The Experts below are selected from a list of 282 Experts worldwide ranked by ideXlab platform

Peter S Mcpherson - One of the best experts on this subject based on the ideXlab platform.

  • dissection of the endogenous cellular pathways of pcsk9 induced low density lipoprotein receptor degradation evidence for an intracellular route
    Journal of Biological Chemistry, 2009
    Co-Authors: Steve Poirier, Peter S Mcpherson, Gaetan Mayer, Viviane Poupon, Roxane Desjardins, Marieclaude Asselin, R Day, Franck Duclos, Mark R Witmer, Rex A Parker
    Abstract:

    Elevated levels of plasma low density lipoprotein (LDL)-cholesterol, leading to familial hypercholesterolemia, are enhanced by mutations in at least three major genes, the LDL receptor (LDLR), its ligand apolipoprotein B, and the proprotein convertase PCSK9. Single point mutations in PCSK9 are associated with either hyper- or hypocholesterolemia. Accordingly, PCSK9 is an attractive target for treatment of dyslipidemia. PCSK9 binds the epidermal growth factor domain A (EGF-A) of the LDLR and directs it to endosomes/lysosomes for destruction. Although the mechanism by which PCSK9 regulates LDLR degradation is not fully resolved, it seems to involve both intracellular and extracellular pathways. Here, we show that Clathrin Light Chain small interfering RNAs that block intracellular trafficking from the trans-Golgi network to lysosomes rapidly increased LDLR levels within HepG2 cells in a PCSK9-dependent fashion without affecting the ability of exogenous PCSK9 to enhance LDLR degradation. In contrast, blocking the extracellular LDLR endocytosis/degradation pathway by a 4-, 6-, or 24-h incubation of cells with Dynasore or an EGF-AB peptide or by knockdown of endogenous autosomal recessive hypercholesterolemia did not significantly affect LDLR levels. The present data from HepG2 cells and mouse primary hepatocytes favor a model whereby depending on the dose and/or incubation period, endogenous PCSK9 enhances the degradation of the LDLR both extra- and intracellularly. Therefore, targeting either pathway, or both, would be an effective method to reduce PCSK9 activity in the treatment of hypercholesterolemia and coronary heart disease.

  • non stoichiometric relationship between Clathrin heavy and Light Chains revealed by quantitative comparative proteomics of Clathrin coated vesicles from brain and liver
    Molecular & Cellular Proteomics, 2005
    Co-Authors: Martine Girard, Peter S Mcpherson, Patrick D Allaire, Francois Blondeau
    Abstract:

    We used tandem mass spectrometry with peptide counts to identify and to determine the relative levels of expression of abundant protein components of highly enriched Clathrin-coated vesicles (CCVs) from rat liver. The stoichiometry of stable protein complexes including Clathrin heavy Chain and Clathrin Light Chain dimers and adaptor protein (AP) heterotetramers was assessed. We detected a deficit of Clathrin Light Chain compared with Clathrin heavy Chain in non-brain tissues, suggesting a level of regulation of Clathrin cage formation specific to brain. The high ratio of AP-1 to AP-2 in liver CCVs is reversed compared with brain where there is more AP-2 than AP-1. Despite this, general endocytic cargo proteins were readily detected in liver but not in brain CCVs, consistent with the previous demonstration that a major function for brain CCVs is recycling synaptic vesicles. Finally we identified 21 CCV-associated proteins in liver not yet characterized in mammals. Our results further validate the peptide accounting approach, reveal new information on the properties of CCVs, and allow for the use of quantitative proteomics to compare abundant components of organelles under different experimental and pathological conditions.

  • huntingtin interacting protein 1 hip1 regulates Clathrin assembly through direct binding to the regulatory region of the Clathrin Light Chain
    Journal of Biological Chemistry, 2005
    Co-Authors: Valerie Legendreguillemin, Martina Metzler, Lu Gan, Jacynthe Philie, Michael R Hayden, Jeanfrancois Lemaire, Peter S Mcpherson
    Abstract:

    Huntingtin interacting protein 1 (HIP1) is a component of Clathrin coats. We previously demonstrated that HIP1 promotes Clathrin assembly through its central helical domain, which binds directly to Clathrin Light Chains (CLCs). To better understand the relationship between CLC binding and Clathrin assembly we sought to dissect this interaction. Using C-terminal deletion constructs of the HIP1 helical domain, we identified a region between residues 450 and 456 that is required for CLC binding. Within this region, point mutations showed the importance of residues Leu-451, Leu-452, and Arg-453. Mutants that fail to bind CLC are unable to promote Clathrin assembly in vitro but still mediate HIP1 homodimerization and heterodimerization with the family member HIP12/HIP1R. Moreover, HIP1 binding to CLC is necessary for HIP1 targeting to Clathrin-coated pits and Clathrin-coated vesicles. Interestingly, HIP1 binds to a highly conserved region of CLC previously demonstrated to regulate Clathrin assembly. These results suggest a role for HIP1/CLC interactions in the regulation of Clathrin assembly.

  • hip1 and hip12 display differential binding to f actin ap2 and Clathrin identification of a novel interaction with Clathrin Light Chain
    Journal of Biological Chemistry, 2002
    Co-Authors: Valerie Legendreguillemin, Martina Metzler, Martine Charbonneau, Lu Gan, Vikramjit Chopra, Jacynthe Philie, Michael R Hayden, Peter S Mcpherson
    Abstract:

    Huntingtin-interacting protein 1 (HIP1) and HIP12 are orthologues of Sla2p, a yeast protein with essential functions in endocytosis and regulation of the actin cytoskeleton. We now report that HIP1 and HIP12 are major components of the Clathrin coat that interact but differ in their ability to bind Clathrin and the Clathrin adaptor AP2. HIP1 contains a Clathrin-box and AP2 consensus-binding sites that display high affinity binding to the terminal domain of the Clathrin heavy Chain and the ear domain of the AP2 alpha subunit, respectively. These consensus sites are poorly conserved in HIP12 and correspondingly, HIP12 does not bind to AP2 nor does it demonstrate high affinity Clathrin binding. Moreover, HIP12 co-sediments with F-actin in contrast to HIP1, which exhibits no interaction with actin in vitro. Despite these differences, both proteins efficiently stimulate Clathrin assembly through their central helical domain. Interestingly, in both HIP1 and HIP12, this domain binds directly to the Clathrin Light Chain. Our data suggest that HIP1 and HIP12 play related yet distinct functional roles in Clathrin-mediated endocytosis.

Frances M Brodsky - One of the best experts on this subject based on the ideXlab platform.

  • Clathrin Light Chain diversity regulates membrane deformation in vitro and synaptic vesicle formation in vivo
    Proceedings of the National Academy of Sciences of the United States of America, 2020
    Co-Authors: Lisa Redlingshofer, Faye Mcleod, Yu Chen, Marine D Camus, Jemima J Burden, Ernest Palomer, Kit Briant, Philip N Dannhauser, Patricia C Salinas, Frances M Brodsky
    Abstract:

    Clathrin Light Chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB, and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal Clathrin function, we characterized the biophysical properties of Clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced Clathrin knee conformation within assemblies, and Clathrin with neuronal CLC mixtures was more effective in membrane deformation than Clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to WT mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highLight differences between the CLC isoforms and show that isoform mixing influences tissue-specific Clathrin activity in neurons, which requires their functional balance.

  • Clathrin Light Chain diversity regulates membrane deformation in vitro and synaptic vesicle formation in vivo
    bioRxiv, 2020
    Co-Authors: Lisa Redlingshofer, Faye Mcleod, Yu Chen, Marine D Camus, Jemima J Burden, Ernest Palomer, Kit Briant, Philip N Dannhauser, Patricia C Salinas, Frances M Brodsky
    Abstract:

    Clathrin Light Chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal Clathrin function, we characterised the biophysical properties of Clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced Clathrin knee conformation within assemblies, and Clathrin with neuronal CLC mixtures was more effective in membrane deformation than Clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to wild-type mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highLight differences between the CLC isoforms and show that isoform mixing influences tissue-specific Clathrin activity in neurons, which requires their functional balance. SIGNIFICANCE STATEMENT This study reveals that diversity of Clathrin Light Chain (CLC) subunits alters Clathrin properties and demonstrates that the two neuronal CLC subunits work together for optimal Clathrin function in synaptic vesicle formation. Our findings establish a role for CLC diversity in synaptic transmission and illustrate how CLC variability expands the complexity of Clathrin to serve tissue-specific functions. ### Competing Interest Statement The authors have declared no competing interest.

  • Clathrin Light Chain diversity regulates budding efficiency in vitro and synaptic vesicle formation in vivo
    bioRxiv, 2020
    Co-Authors: Lisa Redlingshoefer, Faye Mcleod, Yu Chen, Marine D Camus, Jemima J Burden, Ernest Palomer, Kit Briant, Philip N Dannhauser, Patricia C Salinas, Frances M Brodsky
    Abstract:

    Clathrin Light Chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal Clathrin function, we characterised the biophysical properties of Clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced Clathrin knee conformation within assemblies, and Clathrin with neuronal CLC mixtures was more efficient in membrane budding than Clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to wild-type mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highLight functional differences between the CLC isoforms and show that isoform mixing influences tissue-specific Clathrin function in neurons.

  • Clathrin Light Chain a drives selective myosin vi recruitment to Clathrin coated pits under membrane tension
    Nature Communications, 2019
    Co-Authors: Matteo Biancospino, Lisa Redlingshofer, Frances M Brodsky, Gwen R Buel, Carlos Martinez A Nino, Elena Maspero, Rossella Scotto Di Perrotolo, Andrea Raimondi, Janine Weber, Kylie J Walters
    Abstract:

    Clathrin Light Chains (CLCa and CLCb) are major constituents of Clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in Clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of Clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated Clathrin-coated vesicle budding. Clathrin Light Chains (CLCa and CLCb) are major constituents of Clathrin-coated vesicles. Here authors find and structurally characterize the selective interaction between CLCa and the actin motor protein myosin VI which act together to generate the force that leads to invagination and fission at the apical surface.

  • Clathrin Light Chain diversity regulates lattice properties and efficiency of synaptic vesicle formation
    bioRxiv, 2019
    Co-Authors: Lisa Redlingshofer, Faye Mcleod, Yu Chen, Marine D Camus, Jemima J Burden, Kit Briant, Philip N Dannhauser, Patricia C Salinas, Ernest Palomer Vila, Frances M Brodsky
    Abstract:

    Clathrin Light Chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB and both gene products undergo alternative splicing in neurons. To understand how this CLC diversity influences neuronal Clathrin function, we characterised the biophysical properties of Clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC variants differentially influenced Clathrin knee conformation within assemblies, and Clathrin lattices with neuronal CLC mixtures were more effective in membrane bending than those with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice deficient for nCLCa or nCLCb were defective in synaptic vesicle recycling. Mice with only nCLCb had a reduced synaptic vesicle pool compared to wild-type mice, while nCLCa-only mice had increased synaptic vesicle numbers. These findings highLight functional differences between the CLC isoforms and show that isoform mixing influences tissue-specific Clathrin function in neurons.

Sandra K. Lemmon - One of the best experts on this subject based on the ideXlab platform.

  • Clathrin Light Chain directs endocytosis by influencing the binding of the yeast Hip1R homologue, Sla2, to F-actin.
    Molecular Biology of the Cell, 2011
    Co-Authors: Douglas R. Boettner, Brenda J Andrews, Helena Friesen, Sandra K. Lemmon
    Abstract:

    The role of Clathrin Light Chain (CLC) in Clathrin-mediated endocytosis is not completely understood. Previous studies showed that the CLC N-terminus (CLC-NT) binds the Hip1/Hip1R/Sla2 family of membrane/actin–binding factors and that overexpression of the CLC-NT in yeast suppresses endocytic defects of Clathrin heavy-Chain mutants. To elucidate the mechanistic basis for this suppression, we performed synthetic genetic array analysis with a Clathrin CLC-NT deletion mutation (clc1-Δ19-76). clc1-Δ19-76 suppressed the internalization defects of null mutations in three late endocytic factors: amphiphysins (rvs161 and rvs167) and verprolin (vrp1). In actin sedimentation assays, CLC binding to Sla2 inhibited Sla2 interaction with F-actin. Furthermore, clc1-Δ19-76 suppression of the rvs and vrp phenotypes required the Sla2 actin-binding talin-Hip1/R/Sla2 actin-tethering C-terminal homology domain, suggesting that clc1-Δ19-76 promotes internalization by prolonging actin engagement by Sla2. We propose that CLC directs endocytic progression by pruning the Sla2-actin attachments in the Clathrin lattice, providing direction for membrane internalization.

  • novel function of Clathrin Light Chain in promoting endocytic vesicle formation
    Molecular Biology of the Cell, 2006
    Co-Authors: Thomas M Newpher, Fatima Zahra Idrissi, Maria Isabel Geli, Sandra K. Lemmon
    Abstract:

    Clathrin-mediated endocytosis is a major pathway for uptake of lipid and protein cargo at the plasma membrane. The lattices of Clathrin-coated pits and vesicles are comprised of triskelions, each consisting of three oligomerized heavy Chains (HC) bound by a Light Chain (LC). In addition to binding HC, LC interacts with members of the Hip1/R family of endocytic proteins, including the budding yeast homologue, Sla2p. Here, using in vivo analysis in yeast, we provide novel insight into the role of this interaction. We find that overexpression of LC partially restores endocytosis to cells lacking Clathrin HC. This suppression is dependent on the Sla2p binding region of LC. Using live cell imaging techniques to visualize endocytic vesicle formation, we find that the N-terminal Sla2p binding region of LC promotes the progression of arrested Sla2p patches that form in the absence of HC. We propose that LC binding to Sla2p positively regulates Sla2p for efficient endocytic vesicle formation.

  • Novel functions of Clathrin Light Chains: Clathrin heavy Chain trimerization is defective in Light Chain-deficient yeast.
    Journal of Cell Science, 1997
    Co-Authors: Kristen M. Huang, Lisa Gullberg, Karen K. Nelson, Christopher J. Stefan, Kendall J. Blumer, Sandra K. Lemmon
    Abstract:

    Clathrin is a major coat protein involved in sorting and retention of proteins at the late Golgi and in endocytosis from the cell surface. The Clathrin triskelion contains three heavy Chains, which provide the structural backbone of the Clathrin lattice and three Light Chains, which are thought to regulate the formation or disassembly of Clathrin coats. To better understand the function of the Clathrin Light Chain, we characterized yeast strains carrying a disruption of the Clathrin Light Chain gene (CLC1). Light Chain-deficient cells showed phenotypes similar to those displayed by yeast that have a disruption in the Clathrin heavy Chain gene (CHC1). In clc1-delta cells, the steady state level of the Clathrin heavy Chain was reduced to 20%-25% of wild-type levels and most of the heavy Chain was not trimerized. If CHC1 was overexpressed in clc1-delta cells, heavy Chain trimers were detected and several clc1-delta phenotypes were partially rescued. These results indicate that the Light Chain is important for heavy Chain trimerization and the heavy Chain still has some function in the absence of the Light Chain. In yeast, deletion of CHC1 is lethal in strains carrying the scd1-i allele, while strains carrying the scd1-v allele can survive without the heavy Chain. In previous studies we isolated several multicopy suppressors of inviability of chc1-delta scd1-i cells. Surprisingly, one of these suppressors, SCD4, is identical to CLC1. Overexpression of CLC1 in viable chc1-delta scd1-v strains rescued some but not all of the phenotypes displayed by these cells. In the absence of the heavy Chain, the Light Chain was not found in a high molecular mass complex, but still associated with membranes. These results suggest that the Light Chain can function independently of the Clathrin heavy Chain in yeast.

James H Keen - One of the best experts on this subject based on the ideXlab platform.

  • a unique role for Clathrin Light Chain a in cell spreading and migration
    Journal of Cell Science, 2019
    Co-Authors: Oxana M Tsygankova, James H Keen
    Abstract:

    ABSTRACT Clathrin heavy Chain is the structural component of the Clathrin triskelion, but unique functions for the two distinct and highly conserved Clathrin Light Chains (CLCa and CLCb, also known as CLTA and CLTB, respectively) have been elusive. Here, we show that following detachment and replating, CLCa is uniquely responsible for promoting efficient cell spreading and migration. Selective depletion of CLCa, but not of CLCb, reduced the initial phase of isotropic spreading of HeLa, H1299 and HEK293 cells by 60–80% compared to siRNA controls, and wound closure and motility by ∼50%. Surface levels of β1-integrins were unaffected by CLCa depletion. However, CLCa was required for effective targeting of FAK (also known as PTK2) and paxillin to the adherent surface of spreading cells, for integrin-mediated activation of Src, FAK and paxillin, and for maturation of focal adhesions, but not their microtubule-based turnover. Depletion of CLCa also blocked the interaction of Clathrin with the nucleation-promoting factor WAVE complex, and altered actin distribution. Furthermore, preferential recruitment of CLCa to budding protrusions was also observed. These results comprise the first identification of CLCa-specific functions, with implications for normal and neoplastic integrin-based signaling and cell migration.

  • the actin binding protein hip1r associates with Clathrin during early stages of endocytosis and promotes Clathrin assembly in vitro
    Journal of Cell Biology, 2001
    Co-Authors: Asa E Y Engqvistgoldstein, Robin A Warren, James H Keen, Michael M Kessels, John E Heuser, David G Drubin
    Abstract:

    Huntingtin-interacting protein 1 related (Hip1R) is a novel component of Clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to Clathrin, and provide evidence that Hip1R and Clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R–YFP and DsRed–Clathrin Light Chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of ‘unroofed’ cells showed that Hip1R localizes to Clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of Clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes Clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble Clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1R's properties suggest an early endocytic function at the interface between Clathrin, F-actin, and lipids.

  • spatial control of coated pit dynamics in living cells
    Nature Cell Biology, 1999
    Co-Authors: Ibragim Gaidarov, Robin A Warren, Francesca Santini, James H Keen
    Abstract:

    Here we visualize new aspects of the dynamics of endocytotic Clathrin-coated pits and vesicles in mammalian cells by using a fusion protein consisting of green fluorescent protein and Clathrin Light Chain a. Clathrin-coated pits invaginating from the plasma membrane show definite, but highly limited, mobility within the membrane that is relaxed upon treatment with latrunculin B, an inhibitor of actin assembly, indicating that an actin-based framework may be involved in the mobility of these pits. Transient, motile coated vesicles that originate from coated pits can be detected, with multiple vesicles occasionally appearing to emanate from a single pit. Despite their seemingly random distribution, coated pits tend to form repeatedly at defined sites while excluding other regions. This spatial regulation of coated-pit assembly and function is attributable to the attachment of the coated pits to the membrane skeleton.

Jennifer Lippincottschwartz - One of the best experts on this subject based on the ideXlab platform.

  • photoactivatable mcherry for high resolution two color fluorescence microscopy
    Nature Methods, 2009
    Co-Authors: Fedor V Subach, Jennifer Lippincottschwartz, George H Patterson, Suliana Manley, Jennifer M Gillette, Vladislav V Verkhusha
    Abstract:

    Improved photoactivatable red fluorescent proteins are generated by including properties desirable for photoactivated localization microscopy (PALM) as selection criteria. The PAmCherry proteins are superior tags for one- and two-color PALM in fixed cells, among other applications. Also in this issue, McKinney et al. present an improved version of the green-to-red EosFP protein. The reliance of modern microscopy techniques on photoactivatable fluorescent proteins prompted development of mCherry variants that are initially dark but become red fluorescent after violet-Light irradiation. Using ensemble and single-molecule characteristics as selection criteria, we developed PAmCherry1 with excitation/emission maxima at 564/595 nm. Compared to other monomeric red photoactivatable proteins, it has faster maturation, better pH stability, faster photoactivation, higher photoactivation contrast and better photostability. Lack of green fluorescence and single-molecule behavior make monomeric PAmCherry1 a preferred tag for two-color diffraction-limited photoactivation imaging and for super-resolution techniques such as one- and two-color photoactivated localization microscopy (PALM). We performed PALM imaging using PAmCherry1-tagged transferrin receptor expressed alone or with photoactivatable GFP–tagged Clathrin Light Chain. Pair correlation and cluster analyses of the resulting PALM images identified ≤200 nm clusters of transferrin receptor and Clathrin Light Chain at ≤25 nm resolution and confirmed the utility of PAmCherry1 as an intracellular probe.

  • coated pit dynamics
    Nature, 1999
    Co-Authors: Jennifer Lippincottschwartz
    Abstract:

    Many membrane proteins are taken up into the cell by the process of Clathrin-mediated endocytosis — the proteins are pinched off in vesicles coated with the protein Clathrin. A study using a fusion protein (green fluorescent protein combined with the Clathrin Light-Chain) now indicates that formation of these coated pits may be coupled to events at the membrane skeleton, possibly through scaffold proteins.

  • cell biology coated pit dynamics
    Nature, 1999
    Co-Authors: Jennifer Lippincottschwartz
    Abstract:

    Many membrane proteins are taken up into the cell by the process of Clathrin-mediated endocytosis — the proteins are pinched off in vesicles coated with the protein Clathrin. A study using a fusion protein (green fluorescent protein combined with the Clathrin Light-Chain) now indicates that formation of these coated pits may be coupled to events at the membrane skeleton, possibly through scaffold proteins.