The Cytoskeleton

Tim Mitchisontimothy_mitchison@hms.harvard.edu

Cytoskeleton lectures

• Intoduction – diverse protein polymers physically organize cells and promote motility

• Polymerization dynamics– Self-assembly only: intermediate filaments

– Self-assembly + NTP hydrolysis: Actin and microtubules

• Nucleation: controlling where and when polymers form• Motor proteins: mechanical work from ATP hydrolysis• Muscles • Cilia and flagella• Cell division

Why should a PhD student be interested in the cytoskeleton?

• The cytoskeleton challenges us to consider problems of spatial and temporal organization at scales of m and seconds, where molecular dynamics turn into life.

• The cytoskeleton plays fundamental roles in biological processes– Spatial organization of the cell, including organelles and signaling pathways. – Force generation for movement inside cells, and tissue morphogenesis

• The cytoskeleton is an often under-appreciated component of the physiology of specialized cell types, and the pathology associated with many diseases.

• Cytoskeleton questions often drive development of novel microscopy and single-molecule biophysics technology (and vice versa!)

• Direct medical applications– Cytoskeleton poteins can be druggable disease targets– Synthetic cell biology will require spatial organization of cells

1 mm

Frog egg dividingEach cycle takes ~20min

~10-3 m

Xenopus laevis early embryo

Ostreococcus tauriHendersen et al 2007

~10-6 m

Whole cell length scales

E coli

~10-6 m

How is the 2nd cleavage plane oriented orthogonal to the 1st?

~1200m

1) centering

2) orientation

How is the 1st cleavage plane positioned so as to accurately bisect the egg?

Cleaving eggs solve simple geometry problems

About how big is a typical human cell?

1) ~1 m

2) ~10 m

3) ~100 m

About how big is a typical protein molecule?

1) ~1 nm

2) ~10 nm

3) ~100 nm

4) ~1 m

About how long does it take for a protein sized molecule to diffuse

across a ~10m cell?

1) ~ 0.01 sec

2) ~ 0.1 sec

3) ~ 10 sec

4) ~ 1000 sec

How can protein-sized molecules organize micron-sized cells?

Organizing cells using molecules

Long protein polymers

Chemical gradients

Mechanical forces on membranes

Chemical or electrochemical waves

These mechanisms scale in different ways with cell size

Protein polymers physically organize cells and promote motility

For the polymer to effectively integrate over space, its ends must have special properties relative to its middle.

Structural polarity might also be very useful

The biochemistry of the polymer must be compatible with cellular time, length and force scales

Actin filaments; actin

Plasma membrane deformation, contraction, migration

Microtubules; tubulin

Mitosis, organelle transport++ + +

-- - -

Intermediate filaments; keratin, vimentin, neurofilaments, others

Also nuclear lamins

Mechanical integrity. Nuclear organization (nuclear lamins)Role in specifying tissue-specific cell function??

Prokaryote cytoskeleton

• Until recently it was thought that bacteria did not have, or need, a cytoskeleton– Small cell size, rigid cell wall no need for internal organization or physical

strength

• This view has been changed by progress on FtsZ (tubulin relative), MreB & ParM (actin relatives), Crescentin (IF-like), etc (many more)

• So far, bacteria have polymers, but not motor proteins• Bacteria cytoskeleton polymers are involved in cell shape, cell division

and DNA segregation – like eukaryotes. But, it is far from clear exactly how they function, and whether eukaryotic analogies hold

Bacterial actins and their diversity. Ozyamak E, Kollman JM, Komeili A. Biochemistry. 2013 Oct 8;52(40):6928-39. Multidimensional view of the bacterial cytoskeleton. Celler K, Koning RI, Koster AJ, van Wezel GP. J Bacteriol. 2013 Apr;195(8):1627-36. Bacterial cytokinesis: From Z ring to divisome. Lutkenhaus J, Pichoff S, Du S.Cytoskeleton (Hoboken). 2012 Oct;69(10):778-90.Evolution of cytomotive filaments: the cytoskeleton from prokaryotes to eukaryotes. Löwe J, Amos LA. Int J Biochem Cell Biol. 2009 Feb;41(2):323-9.

FtsZ (prokaryotic tubulin relative)

Cell division in prokaryotes & chloroplasts, but not mitochondriaNote: function in cell division very different from microtubules!

MreB: an actin-like protein involved in cell wall biosynthesis

MreB-GFP in B Subtilis Confocal imaging

Garner et al Science 333:222

MreB: an actin-like protein involved in cell wall biosynthesis

Plasma membraneCoupling proteinsPrecursor transport proteins

MreB filaments

Cell wall

MreB-GFP in B Subtilis Confocal imaging

Garner et al Science 333:222

Cell wall biosynthesis enzymes

How is the MreB moving?

1)

2)

Unidirectional(Coherent)

Bidirectional(Incoherent)

Why does this matter?

Model for MreB function in organizing cell wall biosynthesis

MreB oligomers

Bacterial cytoskeleton

-Many outstanding fundamental questions

-Excellent research groups at HMS and FAS

-Druggable targets for antibiotics?

Nematode sperm cells

Nematode sperm are ameboid, not flagelated

Major Sperm Protein (MSP)

- MSP is a small protein, unique to nematode sperm cells- MSP polymerization-depolymerization promotes cell crawling- MSP is actin-like in function, but not in structure

-no ATP binding, non-polar polymer

Stewart and Roberts (2005) Adv Protein Chem. 71:383-99.

Why is MSP interesting?MSP polymerization-depolymerization drives amoeboid motility of nematode sperm, that resembles amoeboid motility driven by actin polymerization-depolymerization in many eukaroytes (eg Neutrophils, Dictyostelium)

Similarities in dynamic organization are striking: polymerization at the leading edge, backwards treadmilling of a cross-linked network that can couple to the substrate, depolymerization near the nucleus

Yet, aspects of actin we consider central to this biology (polar filaments, polymerization coupled to ATP hydrolysis) are not present in MSP

This challenges us to consider what aspects of cytoskeleton strucutre and dynamics are truly fundamental to cell migration and cell polarity.

It also challenges us to think about how a cytoskeleton that can drive cell motility and phagocytosis might evolve

Basic Principles In Cytoskeleton Biology

• Self-assembly• Polarity• Polymer assembly: nucleation vs. elongation• Polymer dynamics, critical concentration• Role of NTP hydrolysis: treadmilling and dynamic instability• Filament binding proteins and their diverse functions• Motor proteins: converting chemical energy into mechanical work

Self-assembly

Spontaneous assembly of subunits (typically proteins) to build an ordered structure

The size and shape of the final structure are governed by the shape and interactions of the subunits

Examples of self-assembled structures:Cytoskeleton filaments, virus coats, bacterial flagella, protein machines.Also, pathogenic protein aggregates (Hemoglobin-S polymers, Prions, Alzheimer’s plaques)

The main driving force for protein self-assembly is:

1) Van der Waals interactions2) Electrostatic interactions3) Hydrogen bonds4) Hydrophobic interactions5) All the above

Polarity: the property of having two different ends.

• Filament polarity. – Polar filament. Each asymmetric subunit points the same way. The

two ends are different and the filament lattice has inherent directionality

Polarity: the property of having two different ends.

• Filament polarity. – Polar filament. Each asymmetric subunit points the same way. The

two ends are different and the filament lattice has inherent directionality

– Non-polar filament. Polypeptides are always asymmetric, but they can polymerize into filament where the two ends are the same and the lattice has no inherent directionalty

Polarity: the property of having two different ends.

• Filament polarity. – Polar filament. Each asymmetric subunit points the same way. The two

ends are different and the filament lattice has inherent directionality

– Non-polar filament. Polypeptides are always asymmetric, but they can polymerize into filament where the two ends are the same and the lattice has no inherent directionalty

• Cell Polarity

Migrating fibroblastEpithelial cells

Neuron

Leading edge

Retracting tail

Basal Apical

Dendrites

Axon

Filament polarity: cytoskeleton polymers

• Polar– Actin filaments– (Some) Prokaryotic actin-related filaments (MreB, ParM)– Microtubules– FtsZ (prokaryotic tubulin-related polymer)

• Non-polar– Intermediate filaments, nuclear lamins– Major Sperm protein filaments in nematode sperm

• “Bipolar”– Myosin II filaments (muscle, non-muscle)– Eg5 tetramer (Kinesin involved in mitosis)

Myosin II minifilamentSinard et al (1989) JCB109:1537

Filament polarity: significance?

• Proteins that bind to the side of the filament will point he same way– Only polar filaments can act as

directional tracks for motor proteins

ATP ADP + Pi

Filament polarity: significance?

• Proteins that bind to the side of the filament will point he same way– Only polar filaments can act as

directional tracks for motor proteins

• Different protein surfaces are exposed at the two ends– End-specific nucleation

– End-specific capping

ATP ADP + Pi

+nucleating factor

Measuring polarity

1) Use proteins that stick to the side in one orientation (EM)

Myosin heads

Pointed end

Barbed endActin filament

An analogous method for microtubules is called “hook decoration”

Measuring polarity

1) Use proteins that stick to the side in one orientation (EM)

Myosin heads

Pointed end

Barbed end

2) Use differential polymerization rate

Rhodamine-tubulin, GTP

Minusend

Plusend

Actin filament

An analogous method for microtubules is called “hook decoration”

Measuring polarity

1) Use proteins that stick to the side in one orientation (EM)

Myosin heads

Pointed end

Barbed end

2) Use differential polymerization rate

Rhodamine-tubulin, GTP

Minusend

Plusend

Actin filament

3) Use motor protein

Kinesin + ATP moves towards plus end

Minusend

Plusend

An analogous method for microtubules is called “hook decoration”

Measuring polarity

4) Image a microtubule tip-tracking protein

- +GTP-tubulinGDP-tubulin

Measuring polarity

4) Image a microtubule tip-tracking protein in living cell

- +

The tip-tracking protein EB1 binds preferentially to GTP-tubulin in microtubules

GTP-tubulinGDP-tubulin

- +

EB1 imaging in Xenopus egg extract

60x TIRFAni Nguyen

Filament polarity : microtubules in cells

+

+

+

+

+

+ +

Motile cell eg fibroblast

Microtubule

+ end(Preferred end for subunit addition)

- end -

Filament polarity : microtubules in cells

+

+

+

+

+

+ +

++

+- -

+ + + +

-- --

-

Motile cell eg fibroblast

AxonEpithelial cell

Microtubule

+ end(Preferred end for subunit addition)

- end -

Note: situation more complex in dendrites. Polarity depends on distance from soma, and can be mixed

Filament polarity : vesicle transport

+

+

+

+

+

+ +

+ + + +

-- --

Microtubules direct vesicle trafficking by acting as tracks for motor proteins. Golgi and lysosomes move towards minus ends while secretory vesicles move towards at plus ends

Motile cell eg fibroblast

Epithelial cell

++

+- --

Neuron

Filament polarity : actin filaments in cells

Barbed end (Preferred end for subunit addition)

pointed end

In the leading edge of a migrating cell, actin filaments are organized with their barbed ends pointing forwards. As the membrane protrudes, new actin subunits polymerize onto these barbed ends

Migrating fibroblast

Lamellipodium: thin sheet with dendritic actin

Filopodium: long, thin rod with bundled actin