Dynamin Constriction Is Concerted And Damped by Membrane Friction 

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Dynamin Is An Atypical GTPase

Dynamin is a GTPase: one monomer binds and hydrolyses one molecule of GTP. It is interesting to compare Dynamin to the classical family of GTPases: the small G proteins. These proteins, like the Ras family, are molecular switch: they are inactive when bound to GDP and active when bound to GTP. Small G proteins have a high anity for nucleotide (for Ras proteins, dissociation constant: Kd = 0.1M [1]) but a very low basal GTP hydrolysis activity (for Ras proteins, GTP hydrolysis rate: 3.4 10􀀀4s􀀀1 [1]). To switch from one state to another, small G proteins require the help of other proteins: a guanine nucleotide exchange factor (GEF) catalizes GDP dissociation and a GTPase-activating protein (GAP) catalizes GTP hydrolysis. Compared to G proteins, Dynamin has a higher basal GTPase activity (GTP hydrolysis rate: 8-30 10􀀀3s􀀀1) and a lower anity for nucleotide (dissociation constant: Kd = 2.5M) (see [143, 11] and Fig.4.8). These biochemical features (low nucleotide anity and high hydrolysis rate) are reminiscent of molecular motors which converts the chemical energy of nucleotide.

Polymerization Stimulates GTPase Activity

Dynamin polymerization stimulates GTPase activity [143, 149]. GTPase rate increases up to 10-fold in conditions favoring spontaneous self-assembly: low ionic strength and high concentration of dynamin [149]. When Dynamin polymerizes on a lipid templates, the GTPase rate even increases up to 100-fold [84]. Structurally the BSE is thought to enhance the GTPase rate by inducing conformational changes upon dimerization [19]. However the precise mechanism by which polymerization stimulates GTPase activity is still not understood.

GTPase Activity Modies The Helical Structure

GTP is not required for self-assembly. Indeed Dynamin mutant K44A, which has a very low GTPase activity, self-assembles like wild type [61] and Dynamin polymerization was observed in absence of nucleotide in nanotube extrusion experiments [115]. However GTPase activity modies Dynamin helical structure. Electron microscopy experiments have revealed two types of structural modications:
increase of the helical pitch at constant radius; When dynamin polymerizes on nanorods, the helical pitch increases from 11 2 to 20 3 nm upon nucleotide treatment while the radius remains constant (40 2.5 nm) (see Fig.4.9.A and [132, 87]).
decrease of both pitch and radius;
For Dynamin polymers prepared on negatively charged liposomes made of pure PS, upon nucleotide treatment it was observed that the outer radius decreases from 50 to 40 nm, the helical pitch decreases from 13 to 9 nm and the number of dimers by helical turn goes from 14 to 13 (see Fig.4.9.B and [137, 31, 23]).
It is likely that these dierent observations of helical structures result from dierent lipid templates for Dynamin polymerization. Indeed membrane properties, for instance the bending rigidity modulus , could inuence Dynamin GTPase activity; exactly like membrane curvature inuences Dynamin polymerization. It is also interesting to note that ssion never occurs in the case of nanorods whereas it does for pure PS liposomes under certain conditions discussed later in section 4.4.2.

Is Constriction Leading to Fission?

The cryo-electron microscopy pictures shown in Figure 4.9.B. clearly suggest that constriction occurs during GTP hydrolysis. However constriction does not lead to ssion in all cases. When GTP is added after xation of Dynamin-coated tubes on the EM grid, small vesicles, remainings of tubes’ fragmentation, are observed. In contrast if GTP is added in solution before xation, unbroken constricted tubes are observed [31]. This implies that ssion occurs in vitro only when nanotubes are attached on a surface. Indeed 3D reconstructions combining cryoEM and crystallography data highlight that the lipid nanotube is still intact underneath the constricted Dynamin helix (see Fig.4.11 and [157, 21]). The internal diameter of this lipid tube is 7 nm. This diameter is not small enough to trigger ssion since hemission2 is predicted to happen at a diameter around 4 nm [76]. These reconstructions are based on the structure of GMPPCPbound Dynamin. So one could argue that more constriction could come from GTP hydrolysis in vivo. But unbroken constricted nanotubes with GTP-bound Dynamin have also been observed by cryoEM ([31]). Reconstructions also lack the PRD domain but this domain is predicted to be on the outer part of the helix, and would not aect dramatically the conformational changes on its own. It could nevertheless recruit Dynamin cofactors which could facilitate ssion.
From all these results, it appears that constriction is not enough to lead to ssion. Which mechanism could be involved?

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Does Dynamin Depolymerize Before Fission?

Dynamin depolymerization has been proposed to be the mechamism leading to ssion. Sedimentation experiments show that, after polymerization, Dynamin detaches gradually from lipids
upon GTP treatment: after a few minutes, around 70% of Dynamin is detached from liposomes [31]. More recently cycles of Dynamin assembly and disassembly have been detected in two types of experiments.
SUPER templates: SUPER templates are SUPported bilayers with Excess membrane Reservoir. They are made by incubating silica beads with liposomes [104]. Membrane budding is observed when Dynamin and GTP are injected on these SUPER templates. A patch of Dynamin can be detected by uorescence at the neck of membrane buds. Fluorescence uctuations in this patch have been measured (see Fig.4.12.A. and B. and [103]). These uctuations are interpreted as cycles of polymerization and depolymerization.
patch clamp: Patch clamp experiments on supported bilayers have been designed to measure the radius of a lipid nanotube by conductance measurement (see Fig.4.12.C.). After injection of Dynamin and GTP, uctuations in the conductance were monitored followed by an abrupt drop corresponding to the tube break. These uctuations are interpreted as uctuations of the tube radius corresponding to cycles of Dynamin assembly and disassembly (see Fig.4.12.D. and [9]).

Table of contents :

Résumé
Abstract
Remerciements
Contents
List of Notations
1 General Introduction 
2 Biological Membranes 
2.1 Lipids: The Building Block Of Membranes
2.1.1 Glycerophospholipids
2.1.2 Sphingolipids
2.1.3 Sterols
2.2 Vesicular Trac
2.2.1 General Features Of Vesicular Transport
2.2.2 Clathrin-Mediated Endocytosis
2.2.3 Synaptic Vesicle Recycling
3 Physics Of Membrane 
3.1 Theoretical Description Of A Membrane
3.1.1 Stretching
3.1.2 Shearing
3.1.3 Bending
3.1.4 Canham-Helfrich Theory
3.1.5 Membrane Tension
3.2 Experimental Studies Of Membrane Mechanics
3.2.1 Control Of Membrane Tension With A Micropipette
3.2.2 Nanotube Extrusion
3.3 Physics Of Membrane Fission
3.3.1 Role Of Bending Energy In Fission
3.3.2 Fission Induced By Lipid Phase-Separation
4 The Dynamin Protein 
4.1 Discovery Of A Membrane-Remodelling GTPase
4.1.1 Discovery
4.1.2 Role In Clathrin-Mediated Endocytosis
4.1.3 Dynamin Is Implicated In Several Membrane-Remodelling Processes
4.1.4 Dynamin-Related Proteins
4.2 Dynamin Structure: From Monomer to Helix
4.2.1 Monomer
4.2.2 The Helix
4.3 Interplay Between GTPase Activity And Polymerization
4.3.1 Dynamin Is An Atypical GTPase
4.3.2 Polymerization Stimulates GTPase Activity
4.3.3 GTPase Activity Modies The Helical Structure
4.4 Fission Mechanism(s)
4.4.1 Spring Or Garrote?
4.4.2 Is Constriction Leading to Fission?
4.4.3 Does Dynamin Depolymerize Before Fission?
4.4.4 How Does Membrane Elasticity Inuence Dynamin-Mediated Fission? .
5 Dynamin Constriction Is Concerted And Damped by Membrane Friction 
5.1 Introduction To Article 1
5.2 Article 1:
Deformation of Dynamin Helices Damped by Membrane Friction.
5.3 Summary of Article 1
6 Fission is Regulated by Membrane Shape 
6.1 Introduction to Article 2
6.2 Article 2: Membrane Shape at the Edge of the Dynamin Helix Sets Location and Duration of Fission
6.3 Complementary Experiments
6.3.1 Constriction Radius
6.3.2 Dynamin Mutant Experiments
6.3.3 Dynamin Depolymerization
6.4 Summary of Article 2
7 Discussion 
7.1 Dynamin: a Molecular Motor
7.2 Dierent Fission Mechanisms?
7.2.1 Common Features Between Phase Separation-Induced and Dynamin- Mediated Fission
7.2.2 Predictions For Other Fission Machineries
7.3 Fission Versus Fusion
7.4 In Vitro Fission Versus In Vivo Fission
7.4.1 In Vitro Helix Versus In Vivo Helix
7.4.2 Membrane Requirements For Fission In Vivo
8 Conclusion 
Bibliographie

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