Clathrin-coated pits with long, dynamin-wrapped necks upon expression of a clathrin antisense RNA

Iversen, Tore Geir; Skretting, Grethe; Deurs, Bo van; Sandvig, Kirsten

doi:10.1073/pnas.0534231100

Cited by 70 publications

(64 citation statements)

References 26 publications

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“…These data indicate that siRNA treatment against clathrin led to a modest decrease in the total amount of dynamin per cell ( Figure 2C). The lack of increase in cellular dynamin in response to clathrin depletion is in contrast to a recently published report in another cell line using an alternative approach to reduce the cellular abundance of clathrin (Iversen et al, 2003). A similar analysis indicated that the average AP-2 immunostaining per cell, as well as the total number of AP-2 assemblies showed a modest increase.…”

Section: Targeted Reduction In Clathrin Heavy and Light Chainscontrasting

confidence: 71%

Highly Cooperative Control of Endocytosis by Clathrin

Moskowitz

Yokoyama

Ryan

2005

MBoC

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Clathrin assembles into a dynamic two-dimensional lattice on the plasma membrane where it plays a critical role in endocytosis. To probe the regulation of this process, we used siRNA against clathrin, in combination with single cell assays for transferrin uptake as well as total internal reflection microscopy, to examine how endocytic rates and membrane dynamics depend upon cellular clathrin concentration ([Clathrin]). We find that endocytosis is tightly controlled by [Clathrin] over a very narrow dynamic range such that small changes in [Clathrin] can lead to large changes in endocytic rates, indicative of a highly cooperative process (apparent Hill coefficient, n > 6). The number of clathrin assemblies at the cell surface was invariant over a wide range of [Clathrin]; however, both the amount of clathrin in each assembly and the subsequent membrane dynamics were steeply dependent on [Clathrin]. Thus clathrin controls the structural dynamics of membrane internalization via a strongly cooperative process. We used this analysis to show that one important regulator of endocytosis, the actin cytoskeleton, acts noncompetitively as a modulator of clathrin function.

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Section: Targeted Reduction In Clathrin Heavy and Light Chainscontrasting

confidence: 71%

Highly Cooperative Control of Endocytosis by Clathrin

Moskowitz

Yokoyama

Ryan

2005

MBoC

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“…Interestingly, the expression level of clathrin heavy chain was found to be downregulated both at the mRNA and protein levels. The concomitant increase of dynamin and decrease of clathrin expression may be explained by the recent results from Iversen et al (2003) showing that a downregulation of clathrin induced an upregulation of dynamin in fibroblasts. These modulations resulted in an accumulation of clathrin-coated pits at the cell surface connected to the plasma membrane via long dynaminwrapped necks (Iversen et al, 2003).…”

Section: Discussionmentioning

confidence: 94%

“…The concomitant increase of dynamin and decrease of clathrin expression may be explained by the recent results from Iversen et al (2003) showing that a downregulation of clathrin induced an upregulation of dynamin in fibroblasts. These modulations resulted in an accumulation of clathrin-coated pits at the cell surface connected to the plasma membrane via long dynaminwrapped necks (Iversen et al, 2003). Taken together, these results suggest that chronic morphine treatment alters membrane and probably receptors trafficking in rat striatum.…”

Section: Discussionmentioning

confidence: 94%

Cytoskeletal Genes Regulation by Chronic Morphine Treatment in Rat Striatum

Marie-Claire

Courtin

Roques

et al. 2004

Neuropsychopharmacol

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It has been previously suggested that morphine can regulate the expression and function of some proteins of the cytoskeleton. In the present study, we used real-time quantitative polymerase chain reaction to examine the effects of chronic morphine administration, in rat striatum, on 14 proteins involved in microtubule polymerization and stabilization, intracellular trafficking, and serving as markers of neuronal growth and degeneration. Chronic morphine treatment led to modulation of the mRNA level of seven of the 14 genes tested. Glial fibrillary acidic protein (Gfap) and activity-regulated cytoskeleton-associated protein (Arc) mRNA were upregulated, while growth associated protein (Gap43), clathrin heavy chain (Cltc), a-tubulin, Tau, and stathmin were downregulated. In order to determine if the regulation of an mRNA correlates with a modulation of the expression of the corresponding protein, immunoblot analyses were performed. With the exception of Gap43, the levels of Cltc, Gfap, Tau, stathmin, and a-tubulin proteins were found to be in good agreement with those from mRNA quantification. These results demonstrate that neuroadaptation to chronic morphine administration in rat striatum implies modifications of the expression pattern of several genes and proteins of the cytoskeleton and cytoskeletonassociated components.

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“…Self-assembly can stimulate dynamin GTPase activity as much as 100-fold (6). Dynamin spirals similar in dimensions to those formed in vitro can be detected as electron dense collars around the elongated necks of endocytic intermediates that accumulate in mammalian cells when clathrinmediated endocytosis is inhibited by perturbing dynamin (7) or clathrin (8) function. Recently, it has been shown that overexpression of an assembly-defective dynamin mutant blocks clathrin-mediated endocytosis (9), confirming that dynamin self-assembly is required.…”

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confidence: 96%

Dynamin GTPase Domain Mutants That Differentially Affect GTP Binding, GTP Hydrolysis, and Clathrin-mediated Endocytosis

Song

Leonard

Schmid

2004

Journal of Biological Chemistry

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The GTPase dynamin is essential for clathrin-mediated endocytosis. Unlike most GTPases, dynamin has a low affinity for nucleotide, a high rate of GTP hydrolysis, and can self-assemble, forming higher order structures such as rings and spirals that exhibit up to 100-fold stimulated GTPase activity. The role(s) of GTP binding and/or hydrolysis in endocytosis remain unclear because mutations in the GTPase domain so far studied impair both. We generated a new series of GTPase domain mutants to probe the mechanism of GTP hydrolysis and to further test the role of GTP binding and/or hydrolysis in endocytosis. Each of the mutations had parallel effects on assembly-stimulated and basal GTPase activities. In contrast to previous reports, we find that mutation of Thr-65 to Ala (or Asp or His) dramatically lowered both the rate of assembly-stimulated GTP hydrolysis and the affinity for GTP. The assemblystimulated rate of hydrolysis was lowered by the mutation of Ser-61 to Asp and increased by the mutation of Thr-141 to Ala without significantly altering the K m for GTP. For some mutants and to a lesser extent for WT dynamin, self-assembly dramatically altered the K m for GTP, suggesting that conformational changes in the active site accompany self-assembly. Analysis of transferrin endocytosis rates in cells overexpressing mutant dynamins revealed a stronger correlation with both the basal and assembly-stimulated rates of GTP hydrolysis than with the calculated ratio of dynamin-GTP/free dynamin, suggesting that GTP binding is not sufficient, and GTP hydrolysis is required for clathrin-mediated endocytosis in vivo.

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Clathrin-coated pits with long, dynamin-wrapped necks upon expression of a clathrin antisense RNA

Cited by 70 publications

References 26 publications

Highly Cooperative Control of Endocytosis by Clathrin

Highly Cooperative Control of Endocytosis by Clathrin

Cytoskeletal Genes Regulation by Chronic Morphine Treatment in Rat Striatum

Dynamin GTPase Domain Mutants That Differentially Affect GTP Binding, GTP Hydrolysis, and Clathrin-mediated Endocytosis

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