GFP-tagged clathrin light chain expressed in Cos cells show many puntae corresponding to clathrin-coated pits

2009 ◽  
Vol 2009 (12) ◽  
pp. pdb.mov69-pdb.mov69
Keyword(s):  
Light Chain ◽  
Coated Pits ◽  
Cos Cells

scholarly journals Light Chain– dependent Regulation of Kinesin's Interaction with Microtubules

1998 ◽  
Vol 143 (4) ◽  
pp. 1053-1066 ◽  
Author(s):  
Kristen J. Verhey ◽  
Donna L. Lizotte ◽  
Tatiana Abramson ◽  
Linda Barenboim ◽  
Bruce J. Schnapp ◽  
...  
Keyword(s):  
Light Chain ◽  
Immunofluorescence Microscopy ◽  
Tail Domain ◽  
Kinesin Light Chain ◽  
Cos Cells ◽  
Sedimentation Behavior ◽  
Cargo Transport ◽  
Stalk Domain ◽  
Heptad Repeats ◽  
Conventional Kinesin

We have investigated the mechanism by which conventional kinesin is prevented from binding to microtubules (MTs) when not transporting cargo. Kinesin heavy chain (HC) was expressed in COS cells either alone or with kinesin light chain (LC). Immunofluorescence microscopy and MT cosedimentation experiments demonstrate that the binding of HC to MTs is inhibited by coexpression of LC. Association between the chains involves the LC NH2-terminal domain, including the heptad repeats, and requires a region of HC that includes the conserved region of the stalk domain and the NH2 terminus of the tail domain. Inhibition of MT binding requires in addition the COOH-terminal 64 amino acids of HC. Interaction between the tail and the motor domains of HC is supported by sedimentation experiments that indicate that kinesin is in a folded conformation. A pH shift from 7.2 to 6.8 releases inhibition of kinesin without changing its sedimentation behavior. Endogenous kinesin in COS cells also shows pH-sensitive inhibition of MT binding. Taken together, our results provide evidence that a function of LC is to keep kinesin in an inactive ground state by inducing an interaction between the tail and motor domains of HC; activation for cargo transport may be triggered by a small conformational change that releases the inhibition of the motor domain for MT binding.

Cloning of Drosophila beta-adaptin and its localization on expression in mammalian cells

1994 ◽  
Vol 107 (3) ◽  
pp. 709-718
Author(s):  
D.R. Camidge ◽  
B.M. Pearse
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Beta Subunits ◽  
Specific Interactions ◽  
Coated Pits ◽  
Cos Cells ◽  
Amino Terminal ◽  
Differential Interaction ◽  
Expression In Mammalian Cells ◽  
Choice Of Partner

A Drosophila cDNA (BAD1) encoding a structural and assembly-competent homologue of the mammalian coated pit beta-adaptins (beta and beta') has been cloned and sequenced. In its amino-terminal region (residues 1–575), the BAD1 sequence appears intermediate between that of the mammalian beta-adaptin and a predicted sequence, from cDNA 105a, which appears to code for a version of beta'-adaptin. To test its functional characteristics, a ‘myc’-tagged version of BAD1 was expressed in Cos cells. The BAD1 protein was detected most clearly in plasma membrane coated pits, where it colocalized with alpha-adaptin, although other coated pits were noted which apparently did not contain alpha-adaptin. However, these are probably gamma-adaptin containing pits, as BAD1 was also found colocalized with gamma-adaptin in Golgi coated pits in which, typically, alpha-adaptin is absent. Immunoprecipitation experiments confirmed that the BAD1 protein was present in both types of adaptor complex, unlike beta-adaptin which complexes with alpha-adaptin and beta'-adaptin which partners gamma-adaptin exclusively. In spite of this, BAD1 expression does not appear to mix alpha-adaptin and gamma-adaptin distribution amongst all the coated pits: thus the location of these adaptor complexes in mammalian cells does not depend on the differences between beta subunits but rather on membrane-specific interactions of other adaptor polypeptides. The differential interaction of beta with alpha-adaptin and beta' with gamma-adaptin in mammalian cells is likely to depend on the few non-conservative differences between their respective sequences and BAD1. Four of these (one with respect to beta and three versus 105a) are clustered in a particular region (residues 155 to 305), which may therefore represent a domain that influences the choice of partner adaptin.

scholarly journals Amphiphysin Heterodimers: Potential Role in Clathrin-mediated Endocytosis

1997 ◽  
Vol 8 (10) ◽  
pp. 2003-2015 ◽  
Author(s):  
Patrick Wigge ◽  
Katinka Köhler ◽  
Yvonne Vallis ◽  
Christopher A. Doyle ◽  
David Owen ◽  
...  
Keyword(s):  
Nerve Terminals ◽  
Coated Pits ◽  
Cos Cells ◽  
Src Homology 3 ◽  
Brain Extracts ◽  
Src Homology 3 Domain ◽  
Src Homology ◽  
Transferrin Uptake ◽  
The Brain

Amphiphysin (Amph) is a src homology 3 domain-containing protein that has been implicated in synaptic vesicle endocytosis as a result of its interaction with dynamin. In a screen for novel members of the amphiphysin family, we identified Amph2, an isoform 49% identical to the previously characterized Amph1 protein. The subcellular distribution of this isoform parallels Amph1, both being enriched in nerve terminals. Like Amph1, a role in endocytosis at the nerve terminal is supported by the rapid dephosphorylation of Amph2 on depolarization. Importantly, the two isoforms can be coimmunoprecipitated from the brain as an equimolar complex, suggesting that the two isoforms act in concert. As determined by cross-linking of brain extracts, the Amph1–Amph2 complex is a 220- to 250-kDa heterodimer. COS cells transfected with either Amph1 or Amph2 show greatly reduced transferrin uptake, but coexpression of the two proteins rescues this defect, supporting a role for the heterodimer in clathrin-mediated endocytosis. Although the src homology 3 domains of both isoforms interact with dynamin, the heterodimer can associate with multiple dynamin molecules in vitro and activates dynamin’s GTPase activity. We propose that it is an amphiphysin heterodimer that drives the recruitment of dynamin to clathrin-coated pits in endocytosing nerve terminals.

scholarly journals Cargo regulates clathrin-coated pit invagination via clathrin light chain phosphorylation

2018 ◽  
Vol 217 (12) ◽  
pp. 4253-4266 ◽  
Author(s):  
Hannes Maib ◽  
Filipe Ferreira ◽  
Stéphane Vassilopoulos ◽  
Elizabeth Smythe
Keyword(s):  
G Protein ◽  
Light Chain ◽  
Transferrin Receptor ◽  
G Protein Coupled Receptors ◽  
Light Chains ◽  
Coated Pits ◽  
Selective Uptake ◽  
Site Specific ◽  
Specific Manner ◽  
G Protein Coupled

Clathrin light chains (CLCs) control selective uptake of a range of G protein–coupled receptors (GPCRs), although the mechanism by which this occurs has remained elusive thus far. In particular, site-specific phosphorylation of CLCb controls the uptake of the purinergic GPCR P2Y12, but it is dispensable for the constitutive uptake of the transferrin receptor (TfR). We demonstrate that phosphorylation of CLCb is required for the maturation of clathrin-coated pits (CCPs) through the transition of flat lattices into invaginated buds. This transition is dependent on efficient clathrin exchange regulated by CLCb phosphorylation and mediated through auxilin. Strikingly, this rearrangement is required for the uptake of P2Y12 but not TfR. These findings link auxilin-mediated clathrin exchange to early stages of CCP invagination in a cargo-specific manner. This supports a model in which CCPs invaginate with variable modes of curvature depending on the cargo they incorporate.

scholarly journals Fast scanned widefield scheme provides tunable and uniform illumination for optimized SMLM on large fields of view

2020 ◽  
Author(s):  
Adrien Mau ◽  
Karoline Friedl ◽  
Christophe Leterrier ◽  
Nicolas Bourg ◽  
Sandrine Lévêque-Fort
Keyword(s):  
Single Molecule ◽  
Total Internal Reflection ◽  
Field Of View ◽  
Uniform Illumination ◽  
Optical Sectioning ◽  
Coated Pits ◽  
Uniform Size ◽  
Cos Cells ◽  
Quantitative Analyses ◽  
Single Molecule Localization Microscopy

AbstractQuantitative analyses in classical fluorescence microscopy and Single Molecule Localization Microscopy (SMLM) require uniform illumination over the field of view; ideally coupled with optical sectioning techniques such as Total Internal Reflection Fluorescence (TIRF) to remove out of focus background. In SMLM, high irradiances (several kW/cm²) are crucial to drive the densely labeled sample into the single molecule regime, and conventional gaussian-shaped lasers will typically restrain the usable field of view to around 40 µm x 40 µm. Here we present Adaptable Scanning for Tunable Excitation Regions (ASTER), a novel and versatile illumination technique that generates uniform illumination over adaptable fields of view and is compatible with illumination schemes from epifluorescence to speckle-free TIRF. For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power, providing constant precision and higher throughput over fields of view 25 times larger than typical. This allows improved clustering analysis and uniform size measurements on sub-100 nm objects, as we demonstrate by imaging nanorulers, microtubules and clathrin-coated pits in COS cells, as well as periodic β2-spectrin along the axons of neurons. ASTER’s sharp, quantitative TIRF and SMLM images up to 200 µm x 200 µm in size pave the way for high-throughput quantification of cellular structures and processes.

5-HT and cAMP induce the formation of coated pits and vesicles and increase the expression of clathrin light chain in sensory neurons of aplysia

Neuron ◽  
1993 ◽  
Vol 10 (5) ◽  
pp. 921-929 ◽  
Author(s):  
Yinghe Hu ◽  
Ari Barzilai ◽  
Mary Chen ◽  
Craig H. Bailey ◽  
Eric R. Kandel
Keyword(s):  
Sensory Neurons ◽  
Light Chain ◽  
Coated Pits

scholarly journals Novel Function of Clathrin Light Chain in Promoting Endocytic Vesicle Formation

2006 ◽  
Vol 17 (10) ◽  
pp. 4343-4352 ◽  
Author(s):  
Thomas M. Newpher ◽  
Fatima-Zahra Idrissi ◽  
Maria Isabel Geli ◽  
Sandra K. Lemmon
Keyword(s):  
Light Chain ◽  
Imaging Techniques ◽  
Vesicle Formation ◽  
Endocytic Vesicle ◽  
Binding Region ◽  
Coated Pits ◽  
Major Pathway ◽  
Heavy Chains ◽  
In Vivo Analysis

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.

Changes Occur in Plasma Membrane Turnover when K562 Leukemia Cells are Induced to Differentiate

1982 ◽  
Vol 40 ◽  
pp. 286-287
Author(s):  
L. M. Marshall
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Intracellular Transport ◽  
Parent Cell ◽  
Membrane Turnover ◽  
Coated Pits ◽  
Surface Distribution ◽  
Specific Proteins ◽  
Erythroleukemic Cell ◽  
Specific Membrane

A human erythroleukemic cell line, metabolically blocked in a late stage of erythropoiesis, becomes capable of differentiation along the normal pathway when grown in the presence of hemin. This process is characterized by hemoglobin synthesis followed by rearrangement of the plasma membrane proteins and culminates in asymmetrical cytokinesis in the absence of nuclear division. A reticulocyte-like cell buds from the nucleus-containing parent cell after erythrocyte specific membrane proteins have been sequestered into its membrane. In this process the parent cell faces two obstacles. First, to organize its erythrocyte specific proteins at one pole of the cell for inclusion in the reticulocyte; second, to reduce or abolish membrane protein turnover since hemoglobin is virtually the only protein being synthesized at this stage. A means of achieving redistribution and cessation of turnover could involve movement of membrane proteins by a directional lipid flow. Generation of a lipid flow towards one pole and accumulation of erythrocyte-specific membrane proteins could be achieved by clathrin coated pits which are implicated in membrane endocytosis, intracellular transport and turnover. In non-differentiating cells, membrane proteins are turned over and are random in surface distribution. If, however, the erythrocyte specific proteins in differentiating cells were excluded from endocytosing coated pits, not only would their turnover cease, but they would also tend to drift towards and collect at the site of endocytosis. This hypothesis requires that different protein species are endocytosed by the coated vesicles in non-differentiating than by differentiating cells.

What do nanometer molecular trajectories tell us about heterogeneous cell surface domaines?

1995 ◽  
Vol 53 ◽  
pp. 786-787
Author(s):  
Watt W. Webb
Keyword(s):  
Cell Surface ◽  
Lipid Membranes ◽  
Surface Proteins ◽  
Protein Diffusion ◽  
Coated Pits ◽  
Cell Surface Proteins ◽  
Membrane Heterogeneity ◽  
Fluorescence Photobleaching Recovery ◽  
Photobleaching Recovery ◽  
Plasma Membrane Heterogeneity

Plasma membrane heterogeneity is implicit in the existence of specialized cell surface organelles which are necessary for cellular function; coated pits, post and pre-synaptic terminals, microvillae, caveolae, tight junctions, focal contacts and endothelial polarization are examples. The persistence of these discrete molecular aggregates depends on localized restraint of the constituent molecules within specific domaines in the cell surface by strong intermolecular bonds and/or anchorage to extended cytoskeleton. The observed plasticity of many of organelles and the dynamical modulation of domaines induced by cellular signaling evidence evanescent intermolecular interactions even in conspicuous aggregates. There is also strong evidence that universal restraints on the mobility of cell surface proteins persist virtually everywhere in cell surfaces, not only in the discrete organelles. Diffusion of cell surface proteins is slowed by several orders of magnitude relative to corresponding protein diffusion coefficients in isolated lipid membranes as has been determined by various ensemble average methods of measurement such as fluorescence photobleaching recovery(FPR).

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