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Biomedical subjects

Sandra L Schmid

Publications and source records attributed to Sandra L Schmid.

At least 19 recordsLinked to original sources
PubMed2006

The dynamin middle domain is critical for tetramerization and higher-order self-assembly.

The large multidomain GTPase dynamin self-assembles around the necks of deeply invaginated coated pits at the plasma membrane and catalyzes vesicle scission by mechanisms that are not yet completely understood. Although a structural role for the 'middle' domain in dynamin function has been suggested, it has not been experimentally established. Furthermore, it is not clear whether this putative function pertains to dynamin structure in the unassembled state or to its higher-order self-assembly or both. Here, we demonstrate that two mutations in this domain, R361S and R399A, disrupt the tetrameric structure of dynamin in the unassembled state and impair its ability to stably bind to and nucleate higher-order self-assembly on membranes. Consequently, these mutations also impair dynamin's assembly-dependent stimulated GTPase activity.

Animals↗
PubMed2006

Domain requirements for an endocytosis-independent, isoform-specific function of dynamin-2.

Endocytosis is inhibited by overexpression of either dynamin-1 or dynamin-2 mutants because both isoforms form heterotetramers with endogenous dynamin-2 and interfere with its function. By contrast, other phenotypes, which are specifically triggered by overexpression of dynamin-2, but not dynamin-1 are likely to reflect endocytosis-independent, dynamin-2-specific functions and/or interactions. Using Dyn2/Dyn1 chimeras, we explored the structural requirements for a readily quantifiable, isoform-specific function of dynamin-2, the activation of caspase-3 to trigger apoptosis. Strikingly, swapping the highly homologous GTPase domain of dynamin-2 into dynamin-1 was sufficient to confer caspase-3 activation. Moreover, assembly-defective mutations in GED, dynamin's GAP/assembly domain, that inhibit endocytosis enhance caspase-3 activation. Thus, this dynamin-2-specific function is mechanistically distinct from and independent of its role in endocytosis. These findings have important implications for interpreting dynamin-2 dependent phenotypes in overexpression studies.

Animals↗
PubMed2006

A cell-free biochemical complementation assay reveals complex and redundant cytosolic requirements for LRP endocytosis.

The low density lipoprotein receptor-related protein (LRP) binds multiple, distinct ligands and participates in constitutive endocytosis and signal transduction. Using an in vitro reconstitution system and a new biochemical complementation assay, we have explored the limiting cytosolic requirements for endocytosis of LRP from isolated plasma membranes. We find that clathrin, AP2 and dynamin do not support efficient LRP uptake and that additional factors present in a 30% ammonium sulfate supernatant fraction of bovine brain cytosol (AS supt) are required. Fractionation of the AS supt revealed that multiple and redundant factors are required to support LRP endocytosis. Among these, we identified Hsc70, synaptojanin1 and CRMP-2 by mass spectrometry. Our data suggest that LRP, which bears several distinct endocytic motifs in its cytoplasmic domain, may use multiple pathways for endocytosis in vitro.

Amino Acid Motifs↗
PubMed2005

Crystal structure of the GTPase domain of rat dynamin 1.

Here, we present the 1.9-A crystal structure of the nucleotide-free GTPase domain of dynamin 1 from Rattus norvegicus. The structure corresponds to an extended form of the canonical GTPase fold observed in Ras proteins. Both nucleotide-binding switch motifs are well resolved, adopting conformations that closely resemble a GTP-bound state not previously observed for nucleotide-free GTPases. Two highly conserved arginines, Arg-66 and Arg-67, greatly restrict the mobility of switch I and are ideally positioned to relay information about the nucleotide state to other parts of the protein. Our results support a model in which switch I residue Arg-59 gates GTP binding in an assembly-dependent manner and the GTPase effector domain functions as an assembly-dependent GTPase activating protein in the fashion of RGS-type GAPs.

Animals↗
PubMed2005

An internal GAP domain negatively regulates presynaptic dynamin in vivo: a two-step model for dynamin function.

The mechanism by which the self-assembling GTPase dynamin functions in vesicle formation remains controversial. Point mutations in shibire, the Drosophila dynamin, cause temperature-sensitive (ts) defects in endocytosis. We show that the ts2 mutation, which occurs in the switch 2 region of dynamin's GTPase domain, compromises GTP binding affinity. Three second-site suppressor mutations, one in the switch 1 region of the GTPase domain and two in the GTPase effector domain (GED), dynamin's putative GAP, fully rescue the shi(ts2) defects in synaptic vesicle recycling. The functional rescue in vivo correlates with a reduction in both the basal and assembly-stimulated GTPase activity in vitro. These findings demonstrate that GED is indeed an internal dynamin GAP and establish that, as for other GTPase superfamily members, dynamin's function in vivo is negatively regulated by its GAP activity. Based on these and other observations, we propose a two-step model for dynamin during vesicle formation in which an early regulatory GTPase-like function precedes late, assembly-dependent steps during which GTP hydrolysis is required for vesicle release.

Amino Acid Sequence↗
PubMed2005

CVAK104 is a novel poly-L-lysine-stimulated kinase that targets the beta2-subunit of AP2.

Isolated clathrin adaptor protein (AP) preparations are known to co-fractionate with endogenous kinase activities, including poly-L-lysine-stimulated kinases that target various constituents of the clathrin coat. We have identified CVAK104 (a coated vesicle-associated kinase of 104 kDa) using a mass spectroscopic analysis of adaptor protein preparations. CVAK104 is a novel serine/threonine kinase that belongs to the SCY1-like family of protein kinases, previously thought to be catalytically inactive. We found that CVAK104 co-fractionates with adaptor protein preparations extracted from clathrin-coated vesicles and directly binds to both clathrin and the plasma membrane adaptor, AP2. CVAK104 binds ATP, and kinase assays indicate that it functions in vitro as a poly-L-lysine-stimulated kinase that is capable of autophosphorylation and phosphorylating the beta2-adaptin subunit of AP2.

Amino Acid Sequence↗
PubMed2005

SNX9 regulates dynamin assembly and is required for efficient clathrin-mediated endocytosis.

Dynamin, a central player in clathrin-mediated endocytosis, interacts with several functionally diverse SH3 domain-containing proteins. However, the role of these interactions with regard to dynamin function is poorly defined. We have investigated a recently identified protein partner of dynamin, SNX9, sorting nexin 9. SNX9 binds directly to both dynamin-1 and dynamin-2. Moreover by stimulating dynamin assembly, SNX9 stimulates dynamin's basal GTPase activity and potentiates assembly-stimulated GTPase activity on liposomes. In fixed cells, we observe that SNX9 partially localizes to clathrin-coated pits. Using total internal reflection fluorescence microscopy in living cells, we detect a transient burst of EGFP-SNX9 recruitment to clathrin-coated pits that occurs during the late stages of vesicle formation and coincides spatially and temporally with a burst of dynamin-mRFP fluorescence. Transferrin internalization is inhibited in HeLa cells after siRNA-mediated knockdown of SNX9. Thus, our results establish that SNX9 is required for efficient clathrin-mediated endocytosis and suggest that it functions to regulate dynamin activity.

Carrier Proteins↗
PubMed2005

Robust colorimetric assays for dynamin's basal and stimulated GTPase activities.

Dynamin, unlike many GTPase superfamily members, exhibits a relatively rapid basal rate of GTP hydrolysis that is not rate-limited by GTP binding or GDP dissociation. Also unique to dynamin GTPase family members is their ability to self-assemble into rings and helical stacks of rings either in solution or onto lipid templates. Self-assembly stimulates dynamin's GTPase activity by >100-fold. Given these robust rates of GTP hydrolysis compared to most GTPases, GTP hydrolysis by dynamin can be easily measured using a simple colorimetic assay to detect released phosphate. We describe this assay and report variations in assay conditions that have contributed to the wide range of reported values for dynamin's basal and assembly-stimulated rates of GTP hydrolysis.

Animals↗
PubMed2005

Clathrin-coated vesicle formation from isolated plasma membranes.

Endocytic clathrin-coated vesicle (CCV) formation is a complex process involving a large number of proteins and lipids. The minimum machinery and the hierarchy of the events involved in CCV formation have yet to be defined. Here we describe an in vitro assay for CCV formation from highly purified rat liver plasma membranes. This rapid and easy assay can be used to quantitatively evaluate the different protein requirements for different endocytic receptors.

Animals↗
PubMed2004

A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis in mammalian cells is critical for a variety of cellular processes including nutrient uptake and cell surface receptor down-regulation. Despite the findings that numerous endocytic accessory proteins directly or indirectly regulate actin dynamics and that actin assembly is spatially and temporally coordinated with endocytosis, direct functional evidence for a role of actin during clathrin-coated vesicle formation is lacking. Here, we take parallel biochemical and microscopic approaches to address the contribution of actin polymerization/depolymerization dynamics to clathrin-mediated endocytosis. When measured using live-cell fluorescence microscopy, disruption of the F-actin assembly and disassembly cycle with latrunculin A or jasplakinolide results in near complete cessation of all aspects of clathrin-coated structure (CCS) dynamics. Stage-specific biochemical assays and quantitative fluorescence and electron microscopic analyses establish that F-actin dynamics are required for multiple distinct stages of clathrin-coated vesicle formation, including coated pit formation, constriction, and internalization. In addition, F-actin dynamics are required for observed diverse CCS behaviors, including splitting of CCSs from larger CCSs, merging of CCSs, and lateral mobility on the cell surface. Our results demonstrate a key role for actin during clathrin-mediated endocytosis in mammalian cells.

3T3 Cells↗
PubMed2004

Dynamin GTPase domain mutants that differentially affect GTP binding, GTP hydrolysis, and clathrin-mediated endocytosis.

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 Km for GTP. For some mutants and to a lesser extent for WT dynamin, self-assembly dramatically altered the Km 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.

Adenoviridae↗
PubMed2004

An assembly-incompetent mutant establishes a requirement for dynamin self-assembly in clathrin-mediated endocytosis in vivo.

Dynamin GTPase activity is required for its biological function in clathrin-mediated endocytosis; however, the role of self-assembly has not been unambiguously established. Indeed, overexpression of a dynamin mutant, Dyn1-K694A, with impaired ability to self-assemble has been shown to stimulate endocytosis in HeLa cells (Sever et al., Nature 1999, 398, 481). To identify new, assembly-incompetent mutants of dynamin 1, we made point mutations in the GTPase effector/assembly domain (GED) and tested for their effects on self-assembly and clathrin-mediated endocytosis. Mutation of three residues, I690, K694, and I697, suggests that interactions with an amphipathic helix in GED are required for self-assembly. In particular, Dyn1-I690K failed to exhibit detectable assembly-stimulated GTPase activity under all assay conditions. Overexpression of this assembly-incompetent mutant inhibited transferrin endocytosis as potently as the GTPase-defective dominant-negative mutant, Dyn1-K44A. However, worm-like endocytic intermediates accumulated in cells expressing Dyn1-I690K that were structurally distinct from long tubules that accumulated in cells expressing Dyn1-K44A. Together these results provide new structural insight into the role of GED in self-assembly and assembly-stimulated GTPase activity and establish that dynamin self-assembly is essential for clathrin-mediated endocytosis.

Amino Acid Sequence↗
PubMed2003

Modulation of Rac localization and function by dynamin.

The GTPase dynamin controls a variety of endocytic pathways, participates in the formation of phagosomes, podosomal adhesions, and invadopodia, and in regulation of the cytoskeleton and apoptosis. Rac, a member of the Rho family of small GTPases, controls formation of lamellipodia and focal complexes, which are critical in cell migration and phagocytosis. We now show that disruption of dynamin(-2) function alters Rac localization and inhibits cell spreading and lamellipodia formation even though Rac is activated. Dominant-negative K44A dynamin(-2) inhibited cell spreading and lamellipodia formation on fibronectin without blocking cell adhesion; dynamin(-2) depletion by specific small interfering RNA inhibited lamellipodia in a similar manner. Dyn2(K44A) induced Rac mislocalization away from cell edges, into abnormal dorsal ruffles, and led to increased total Rac activity. Fluorescence resonance energy transfer imaging of Rac activity confirmed its predominant localization to aberrant dorsal ruffles in the presence of dominant-negative dyn2(K44A). Dyn2(K44A) induced the accumulation of tubulated structures bearing membrane-bound Rac-GFP. Constitutively active but not wild-type GFP-Rac was found on macropinosomes and Rac-dependent, platelet-derived growth factor-induced macropinocytosis was abolished by Dyn2(K44A) expression. These data suggest an indispensable role of dynamin in Rac trafficking to allow for lamellipodia formation and cell spreading.

Animals↗
PubMed2003

Differential requirements for AP-2 in clathrin-mediated endocytosis.

AP-2 complexes are key components in clathrin-mediated endocytosis (CME). They trigger clathrin assembly, interact directly with cargo molecules, and recruit a number of endocytic accessory factors. Adaptor-associated kinase (AAK1), an AP-2 binding partner, modulates AP-2 function by phosphorylating its mu2 subunit. Here, we examined the effects of adenoviral-mediated overexpression of WT AAK1, kinase-dead, and truncation mutants in HeLa cells, and show that AAK1 also regulates AP-2 function in vivo. WT AAK1 overexpression selectively blocks transferrin (Tfn) receptor and LRP endocytosis. Inhibition was kinase independent, but required the full-length AAK1 as truncation mutants were not inhibitory. Although changes in mu2 phosphorylation were not detected, AAK1 overexpression significantly decreased the phosphorylation of large adaptin subunits and the normally punctate AP-2 distribution was dispersed, suggesting that AAK1 overexpression inhibited Tfn endocytosis by functionally sequestering AP-2. Surprisingly, clathrin distribution and EGF uptake were unaffected by AAK1 overexpression. Thus, AP-2 may not be stoichiometrically required for coat assembly, and may have a more cargo-selective function in CME than previously thought.

Adaptor Protein Complex 2↗
PubMed2003

Regulated portals of entry into the cell.

The plasma membrane is the interface between cells and their harsh environment. Uptake of nutrients and all communication among cells and between cells and their environment occurs through this interface. 'Endocytosis' encompasses several diverse mechanisms by which cells internalize macromolecules and particles into transport vesicles derived from the plasma membrane. It controls entry into the cell and has a crucial role in development, the immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. As the complexity of molecular interactions governing endocytosis are revealed, it has become increasingly clear that it is tightly coordinated and coupled with overall cell physiology and thus, must be viewed in a broader context than simple vesicular trafficking.

Actins↗