Ubiquitously Expressed Dynamin-II Has a Higher Intrinsic GTPase Activity and a Greater Propensity for Self-assembly Than Neuronal Dynamin-I

To begin to understand mechanistic differences in endocytosis in neurons and nonneuronal cells, we have compared the biochemical properties of the ubiquitously expressed dynamin-II isoform with those of neuron-specific dynamin-I. Like dynamin-I, dynamin-II is specifically localized to and highly concentrated in coated pits on the plasma membrane and can assemble in vitro into rings and helical arrays. As expected, the two closely related isoforms share a similar mechanism for GTP hydrolysis: both are stimulated in vitro by self-assembly and by interaction with microtubules or the SH3 domain-containing protein, grb2. Deletion of the C-terminal proline/arginine-rich domain from either isoform abrogates self-assembly and assembly-dependent increases in GTP hydrolysis. However, dynamin-II exhibits a ∼threefold higher rate of intrinsic GTP hydrolysis and higher affinity for GTP than dynamin-I. Strikingly, the stimulated GTPase activity of dynamin-II can be >40-fold higher than dynamin-I, due principally to its greater propensity for self-assembly and the increased resistance of assembled dynamin-II to GTP-triggered disassembly. These results are consistent with the hypothesis that self-assembly is a major regulator of dynamin GTPase activity and that the intrinsic rate of GTP hydrolysis reflects a dynamic, GTP-dependent equilibrium of assembly and disassembly.

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Identification and Characterization of the Endocytic Transmembrane Glycoprotein Endo180 as a Novel Collagen Receptor

Molecular Biology of the Cell ◽

10.1091/mbc.e02-12-0814 ◽

2003 ◽

Vol 14 (9) ◽

pp. 3592-3604 ◽

Cited By ~ 98

Author(s):

Dirk Wienke ◽

John R. MacFadyen ◽

Clare M. Isacke

Keyword(s):

Physiological Role ◽

Collagen Matrix ◽

Mannose Receptor ◽

In Vitro Assays ◽

Type Ii ◽

Urokinase Plasminogen Activator Receptor ◽

Coated Pits ◽

Rich Domain ◽

Fibronectin Type

Endo180, a member of the mannose receptor family, is constitutively recycled between clathrin-coated pits on the cell surface and intracellular endosomes. Its large extracellular domain contains an N-terminal cysteine-rich domain, a single fibronectin type II domain and eight C-type lectin-like domains. The second of these lectin-like domains has been shown to mediate Ca2+-dependent mannose binding. In addition, cross-linking studies have identified Endo180 as a urokinase plasminogen activator receptor–associated protein and this interaction can be blocked by collagen V. Here we demonstrate directly using in vitro assays, cell-based studies and tissue immunohistochemistry that Endo180 binds both to native and denatured collagens and provide evidence that this is mediated by the fibronectin type II domain. In cell culture systems, expression of Endo180 results in the rapid uptake of soluble collagens for delivery to lysosomal degradative compartments. Together with the observed restricted expression of Endo180 in both embryonic and adult tissue, we propose that Endo180 plays a physiological role in mediating collagen matrix remodelling during tissue development and homeostasis and that the observed receptor upregulation in pathological conditions may contribute to disease progression.

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Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles

The Journal of Cell Biology ◽

10.1083/jcb.200308110 ◽

2003 ◽

Vol 163 (4) ◽

pp. 813-824 ◽

Cited By ~ 135

Author(s):

Kazuhito Tomizawa ◽

Satoshi Sunada ◽

Yun-Fei Lu ◽

Yoshiya Oda ◽

Masahiro Kinuta ◽

...

Keyword(s):

Hippocampal Neurons ◽

Critical Role ◽

Ring Formation ◽

Free System ◽

Rich Domain ◽

A Cell ◽

Dynamin I ◽

Deficient Mice

It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation–induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with β-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.

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Biophysical and biochemical properties of Deup1 self-assemblies: a potential driver for deuterosome formation during multiciliogenesis

Biology Open ◽

10.1242/bio.056432 ◽

2021 ◽

Vol 10 (3) ◽

pp. bio056432 ◽

Cited By ~ 1

Author(s):

Shohei Yamamoto ◽

Ryoichi Yabuki ◽

Daiju Kitagawa

Keyword(s):

Self Assembly ◽

Large Scale ◽

Molecular Mechanisms ◽

Coiled Coil ◽

Biochemical Properties ◽

The Self ◽

Centrosomal Protein ◽

Biochemical Analyses ◽

Stable Structures

ABSTRACTThe deuterosome is a non-membranous organelle involved in large-scale centriole amplification during multiciliogenesis. Deuterosomes are specifically assembled during the process of multiciliogenesis. However, the molecular mechanisms underlying deuterosome formation are poorly understood. In this study, we investigated the molecular properties of deuterosome protein 1 (Deup1), an essential protein involved in deuterosome assembly. We found that Deup1 has the ability to self-assemble into macromolecular condensates both in vitro and in cells. The Deup1-containing structures formed in multiciliogenesis and the Deup1 condensates self-assembled in vitro showed low turnover of Deup1, suggesting that Deup1 forms highly stable structures. Our biochemical analyses revealed that an increase of the concentration of Deup1 and a crowded molecular environment both facilitate Deup1 self-assembly. The self-assembly of Deup1 relies on its N-terminal region, which contains multiple coiled coil domains. Using an optogenetic approach, we demonstrated that self-assembly and the C-terminal half of Deup1 were sufficient to spatially compartmentalize centrosomal protein 152 (Cep152) and polo like kinase 4 (Plk4), master components for centriole biogenesis, in the cytoplasm. Collectively, the present data suggest that Deup1 forms the structural core of the deuterosome through self-assembly into stable macromolecular condensates.This article has an associated First Person interview with the first author of the paper.

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Assembly properties of bacterial tubulin homolog FtsZ regulated by the positive regulator protein ZipA and ZapA from Pseudomonas aeruginosa

Scientific Reports ◽

10.1038/s41598-020-78431-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Mujeeb ur Rahman ◽

Zhe Li ◽

Tingting Zhang ◽

Shuheng Du ◽

Xueqin Ma ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Species ◽

Biochemical Properties ◽

Gtpase Activity ◽

Bacterial Cell Division ◽

Contractile Ring ◽

Positive Regulator ◽

Regulator Protein ◽

Z Ring

AbstractBacterial tubulin homolog FtsZ self-assembles into dynamic protofilaments, which forms the scaffold for the contractile ring (Z-ring) to achieve bacterial cell division. Here, we study the biochemical properties of FtsZ from Pseudomonas aeruginosa (PaFtsZ) and the effects of its two positive regulator proteins, ZipA and ZapA. Similar to Escherichia coli FtsZ, PaFtsZ had a strong GTPase activity, ~ 7.8 GTP min-1 FtsZ-1 at pH 7.5, and assembled into mainly short single filaments in vitro. However, PaFtsZ protofilaments were mixtures of straight and “intermediate-curved” (100–300 nm diameter) in pH 7.5 solution and formed some bundles in pH 6.5 solution. The effects of ZipA on PaFtsZ assembly varied with pH. In pH 6.5 buffer ZipA induced PaFtsZ to form large bundles. In pH 7.5 buffer PaFtsZ-ZipA protofilaments were not bundled, but ZipA enhanced PaFtsZ assembly and promoted more curved filaments. Comparable to ZapA from other bacterial species, ZapA from P. aeruginosa induced PaFtsZ protofilaments to associate into long straight loose bundles and/or sheets at both pH 6.5 and pH 7.5, which had little effect on the GTPase activity of PaFtsZ. These results provide us further information that ZipA functions as an enhancer of FtsZ curved filaments, while ZapA works as a stabilizer of FtsZ straight filaments.

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A high-throughput assay to identify robust inhibitors of dynamin GTPase activity

10.1101/153106 ◽

2017 ◽

Author(s):

Aparna Mohanakrishnan ◽

Triet Vincent M. Tran ◽

Meera Kumar ◽

Hong Chen ◽

Bruce A. Posner ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Gtpase Activity ◽

Response Curves ◽

Coated Pits ◽

Major Pathway ◽

Gtp Hydrolysis ◽

Extensive Search ◽

Highly Sensitive ◽

High Throughput Assay

AbstractClathrin-mediated endocytosis is the major pathway by which cells internalize materials from the external environment. Dynamin, a large multidomain GTPase, is a key regulator of clathrin-mediated endocytosis. It assembles at the necks of invaginated clathrin-coated pits and, through GTP hydrolysis, catalyzes scission and release of clathrin-coated vesicles from the plasma membrane. Several small molecule inhibitors of dynamin’s GTPase activity, such as Dynasore and Dyngo-4a, are currently available, although their specificity has been brought into question. Previous screens for these inhibitors measured dynamin’s stimulated GTPase activity due to lack of sufficient sensitivity, hence the mechanisms by which they inhibit dynamin are uncertain. We report a highly sensitive fluorescence-based assay capable of detecting dynamin’s basal GTPase activity under conditions compatible with high throughput screening. Utilizing this optimized assay, we conducted a pilot screen of 8000 compounds and identified several “hits” that inhibit the basal GTPase activity of dynamin-1. Subsequent dose-response curves were used to validate the activity of these compounds. Interestingly, we found neither Dynasore nor Dyngo-4a inhibited dynamin’s basal GTPase activity, although both inhibit assembly-stimulated GTPase activity. This assay provides the basis for a more extensive search for robust dynamin inhibitors.

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Dynamin:Gtp Controls the Formation of Constricted Coated Pits, the Rate Limiting Step in Clathrin-Mediated Endocytosis

The Journal of Cell Biology ◽

10.1083/jcb.150.5.1137 ◽

2000 ◽

Vol 150 (5) ◽

pp. 1137-1148 ◽

Cited By ~ 166

Author(s):

Sanja Sever ◽

Hanna Damke ◽

Sandra L. Schmid

Keyword(s):

Self Assembly ◽

Point Mutations ◽

Coated Vesicle ◽

Primary Defect ◽

Coated Pits ◽

Gtp Hydrolysis ◽

Receptor Mediated Endocytosis ◽

Rate Limiting Step ◽

Limiting Step ◽

Rate Limiting

The GTPase dynamin is essential for receptor-mediated endocytosis, but its function remains controversial. A domain of dynamin, termed the GTPase effector domain (GED), controls dynamin's high stimulated rates of GTP hydrolysis by functioning as an assembly-dependent GAP. Dyn(K694A) and dyn(R725A) carry point mutations within GED resulting in reduced assembly stimulated GTPase activity. Biotinylated transferrin is more rapidly sequestered from avidin in cells transiently overexpressing either of these two activating mutants (Sever, S., A.B. Muhlberg, and S.L. Schmid. 1999. Nature. 398:481–486), suggesting that early events in receptor-mediated endocytosis are accelerated. Using stage-specific assays and morphological analyses of stably transformed cells, we have identified which events in clathrin-coated vesicle formation are accelerated by the overexpression of dyn(K694A) and dyn(R725A). Both mutants accelerate the formation of constricted coated pits, which we identify as the rate limiting step in endocytosis. Surprisingly, overexpression of dyn(R725A), whose primary defect is in stimulated GTP hydrolysis, but not dyn(K694A), whose primary defect is in self-assembly, inhibited membrane fission leading to coated vesicle release. Together, our data support a model in which dynamin functions like a classical GTPase as a key regulator of clathrin-mediated endocytosis.

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Differential regulation of cellular activities by GTPase-activating protein and NF1.

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2497 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2497-2503 ◽

Cited By ~ 19

Author(s):

N al-Alawi ◽

G Xu ◽

R White ◽

R Clark ◽

F McCormick ◽

...

Keyword(s):

Signal Transduction ◽

Biological Effects ◽

Type I ◽

Type Ii ◽

Gtpase Activity ◽

Ras Proteins ◽

Ras Gtpase ◽

Intrinsic Gtpase Activity

The regulation of the GTPase activity of the Ras proteins is thought to be a key element of signal transduction. Ras proteins have intrinsic GTPase activity and are active in signal transduction when bound to GTP but not following hydrolysis of GTP to GDP. Three cellular Ras GTPase-activating proteins (Ras-gaps) which increase the GTPase activity of wild-type (wt) Ras but not activated Ras in vitro have been identified: type I and type II GAP and type I NF1. Mutations of wt Ras resulting in lowered intrinsic GTPase activity or loss of response to cellular Ras-gap proteins are thought to be the primary reason for the transforming properties of the Ras proteins. In vitro assays show type I and type II GAP and the GAP-related domain of type I NF1 to have similar biochemical properties with respect to activation of the wt Ras GTPase, and it appears as though both type I GAP and NF1 can modulate the GTPase function of Ras in cells. Here we report the assembling of a full-length coding clone for type I NF1 and the biological effects of microinjection of Ras and Ras-gap proteins into fibroblasts. We have found that type I GAP, type II GAP, and type I NF1 show markedly different biological activities in vivo. Coinjection of type I GAP or type I NF1, but not type II GAP, with wt Ras abolished the ability of wt Ras to induce expression from an AP-1-controlled reporter gene. We also found that serum-stimulated DNA synthesis was reduced by prior injection of cells with type I GAP but not type II GAP or type I NF1. These results suggest that type I GAP, type II GAP, and type I NF1 may have different activities in vivo and support the hypothesis that while type I forms of GAP and NF1 may act as negative regulators of wt Ras, they may do so with differential efficiencies.

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Self-Assembly of Dynamin

Microscopy and Microanalysis ◽

10.1017/s1431927600032128 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 1210-1211

Author(s):

Dganit Danino ◽

Jenny E. Hinshaw

Keyword(s):

Plasma Membrane ◽

Self Assembly ◽

Wild Type ◽

Salt Solutions ◽

Coated Pits ◽

Vesicle Recycling ◽

Rich Domain ◽

Gtp Binding ◽

Low Salt ◽

Over Time

Dynamin is a large GTPase essential for various intracellular processes such as synaptic vesicle recycling, caveolae internalization and trafficking into and out of the Golgi. It is also involved in receptor-mediated endocytosis, and is believed to assemble at the necks of clathrin-coated pits and assist in pinching vesicles from the plasma membrane upon GTP binding and hydrolysis.Purified recombinant dynamin self-assembles into rings and spirals in low salt conditions [1]. A dynamin mutant lacking the c-terminal proline rich domain (APRD) also assembles into rings and spirals, however unlike wild type dynamin APRD constricts in the presence of GTP analogous such as GMP-PCP [2] or GTPγS. to explore differences in the behavior of the wild type and mutant dynamin we dialyzed them into different salt solutions containing various types of nucleotides and studied their assembly over time using negative staining and cryo-TEM.

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In vitro assembly and GTP hydrolysis by bacterial tubulins BtubA and BtubB

The Journal of Cell Biology ◽

10.1083/jcb.200410027 ◽

2005 ◽

Vol 169 (2) ◽

pp. 233-238 ◽

Cited By ~ 59

Author(s):

Christopher A. Sontag ◽

James T. Staley ◽

Harold P. Erickson

Keyword(s):

Critical Concentration ◽

Three Dimensional ◽

Outer Diameter ◽

Gtpase Activity ◽

Gtp Hydrolysis ◽

Assembly Mechanism ◽

In Vitro Assembly ◽

Cooperative Assembly ◽

Versus Concentration

Arecent study identified genuine tubulin proteins, BtubA and BtubB, in the bacterial genus Prosthecobacter. We have expressed BtubA and BtubB in Escherichia coli and studied their in vitro assembly. BtubB by itself formed rings with an outer diameter of 35–36 nm in the presence of GTP or GDP. Mixtures of BtubB and BtubA formed long protofilament bundles, 4–7 protofilaments wide (20–30 protofilaments in the three-dimensional bundle). Regardless of the starting stoichiometry, the polymers always contained equal concentrations of BtubA and BtubB, suggesting that BtubA and B alternate along the protofilament. BtubA showed negligible GTP hydrolysis, whereas BtubB hydrolyzed 0.40 mol GTP per min per mol BtubB. This GTPase activity increased to 1.37 per min when mixed 1:1 with BtubA. A critical concentration of 0.4–1.0 μM was indicated by light scattering experiments and extrapolation of GTPase versus concentration, thus suggesting a cooperative assembly mechanism.

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FtsZ induces membrane deformations via torsional stress upon GTP hydrolysis

Nature Communications ◽

10.1038/s41467-021-23387-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Diego A. Ramirez-Diaz ◽

Adrián Merino-Salomón ◽

Fabian Meyer ◽

Michael Heymann ◽

Germán Rivas ◽

...

Keyword(s):

Optical Tweezers ◽

Lipid Vesicles ◽

Dependent Manner ◽

Torsional Stress ◽

Gtpase Activity ◽

Giant Vesicles ◽

Gtp Hydrolysis ◽

Vitro Assay ◽

Membrane Deformations

AbstractFtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far, however, not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we design an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ filaments actively transform these tubes into spring-like structures, where GTPase activity promotes spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, the directional forces that FtsZ-YFP-mts rings exert upon GTP hydrolysis can be estimated to be in the pN range. They are sufficient to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. We hypothesize that these forces result from torsional stress in a GTPase activity dependent manner.

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