Drunken sailors and sober cells, the ways they move

Shashank ShekharBrandeis University

Cell motility is conserved across billions of years of evolution

White blood cells(Slightly speeded up)David Rogers, 1950

Blood smear

Fish scale cell(30 X speeded up)

Theriot labWound healing

Amoeba(Real time)

Feeds on bacteria in soil

100,000 years ago 2 billion years ago 500 million years ago

Bacteria White blood cell

5 µm 5 µm 25µm

"Run and Tumble" motility of Listeria Monocytogenes

< 30oC

Gründling et al, 2004

1 µm

~ 37oCListeria’s “Run and Tumble”

1 µm

5 µm

Intracellular pathogens also show similiar behaviour

Listeria BacteriaTheriot lab (60X speeded up)

Comet tail

Speedboat

Contrails

Cellular and pathogenic motility have the same molecular basis

Cameron et al, 2001Svitkina et al 1997

1. Similar rates of propulsion (~µm/min)2. Suggests identical molecular basis.3. Evolutionary conserved

Fish scale cell Pathogen

Bacterial and cellular motility have the same molecular basis

Svitkina et al 1997; Gary Borisy Cameron et al, 2001

Actin filament

Actin filaments are formed from monomers

5 nm

Pollard and Cooper, 2001

How do such small molecules self-assemble to form structures orders of magnitude bigger

Fluorescence microscopy enables direct visualization of actin filaments

Actin monomer

Fluorophore

Fluorescence microscopy enables direct visualization of actin filaments

• Filament growth• Thermal fluctuations

Flow

5 µm Flow

Actin polymerization can generate force

Displacement

Short filament (10 µm)

Fixed surface

5 pN

Buckling

Filament networks enable overcoming buckling

Productive

NonProductive

Listeria

Requirements for actin propulsion

Polymerization = force Filament mesh prevents buckling

Elongate productive Filaments Regeneration of monomers

Approach : What I can not create, I don’t understand.

1. Identify essential components2. Characterize the component’s function3. Reconstitute function i.e. motility

Requirements for actin propulsion

Polymerization = force Filament mesh prevents buckling

“Branchers” branch actin filaments to form a dense mesh

Svitkina & Borisy, 1999

Mullins et al., 1999

Actin

Brancher(Arp2/3)

binding elongationArp2/3Filament

dissociation

Smith et al, 2013; Gelles and Goode la

5 µm

Requirement 1 : Generating a dense mesh of filaments

Dense branching forms a mesh

Wiesner et al, 2003

Actin Brancher

Actin

Brancher

Listeria mimicking bead

Listeria placed in mix of Actin + ArpGel formed

Requirement 1 : Branching generates a dense mesh of filaments

Bacteria (Listeria)

Actin

Branchers

Actin

Requirements for actin propulsion

Polymerization = force Filament mesh prevents buckling

Elongate productive Filaments

Requirement 2 : Blocking unproductive growth

5 µm

54 min

Time

Leng

th

Flow

Requirement 2 : Absence of capping leads to a “fish bone” comet

Bacteria (Listeria)

Cappers

Branchers

Actin

Actin + Brancher + Cappers

Carlier et al, 2000

Requirements for actin propulsion

Polymerization = force Filament mesh prevents buckling

Elongate productive Filaments Regeneration of monomers

Requirement 3 : Disassembly of the network & monomer regeneration

Requirement 3 : Disassembly of the network & monomer regeneration

Fast (10 monomers/µM/s)

Slow (0.2 monomers/s)

5 Plus ends

1 minus ends

Debrancher – GMFActin Depolymerizing Factor (ADF)

Requirement 3 : Debrancher increases the number of minus ends

DebrancherGandhi et al, 2010; Sean Guo; Goode and Gelles labs

Requirement 3 : Disassembly of the network & monomer regeneration

5

5

5

1

Debrancher

Plus ends

Minus ends

Requirement 3 : Depolymerizer fragments actin filaments

Actin + Depolymerizer

5 µm

Silvia Jansen

Requirement 3 : Depolymerizer also depolymerizes filaments

Depolymerization increases 20x

0 1 2 3 4 50

1

2

3

4

5

Rat

e of

dep

olym

eriz

atio

n (s

u/s)

ADF (µM)

Depo

lym

eriza

tion

Subu

nits

/sec

Depolymerizer(µM)

Shekhar et al, 2017

Requirement 3 : Depolymerizer fragments and depolymerizes filaments

FastDepolymerization

Shekhar et al, 2017

Requirement 3 : Disassembly of the network & monomer regeneration

Depolymerizer

Summary

1. Actin monomers polymerize to form filaments.

2. Branchers branch filaments.

3. Cappers block filament growth.

4. Depolymerizers depolymerize.

5. Bacteria (Listeria)

Reconstitution of Actin based motility

Loisel et al, 1999

Reconstitution of Actin Based Motility

Listeria

Branching activator (ActA)

Reconstitution of Actin Based Motility

Achard et al, 2010

Delatour et al, 2008

Wiesner et al, 2003

5 µm

Truly an international and interdisciplinary effort

Jané Kondev

Bruce Goode

Jeff Gelles

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