An introduction to Molecular Motors: from single molecules to function in the cell Steven Gross...

An introduction to Molecular Motors: from single molecules to function in the cell

Steven GrossDepartment of Developmental and Cell Biology & Department of

PhysicsUniversity of California, Irvine

Korean Institute for Advanced Study (KIAS), Seoul

Website: bioweb.bio.uci.edu/sgross/

D103 Cell Biology –SS1 2009 Lecture 1+2 © Grün - all rights reserved.

Cell Biology: a brief intro

Cos-7 cells © Nikon MicroscopyU 2

tThanks to Changbong & the other organizers!


Introduction to Cell Biology

I. Introduction to cell biology• Why study cell biology?

…..biology can guide physics• What is the biology trying to achieve?

II. General principles• All cells are prokaryotic or eukaryotic• Investigating cells:

Cellular organization

Role of motors

3Summer School– lecture 1 © Gross - all rights reserved.


"There is a paradox in the growth of scientific knowledge. As information accumulates in ever more intimidating quantities, disconnected facts and impenetrable mysteries give way to rational explanations, and simplicity emerges from chaos.”

Alberts, et al.

• Fascinating

• Provides insight into the mechanism of how cells work We have to understand the details of a single cell in order to understand a complex, multi-cellular organism.

• Essential for the understanding and the treatment of human diseases.

Why Study Cell Biology ?

4Summer School– lecture 1 © Gross - all rights reserved.


Prokaryotic cell=Bacteria• Simpler organization than Eukaryotic cell• Some systems similar to eukaryotic cell

All cells are prokaryotic or

eukaryotic

5lecture 1 © Gross - all rights reserved.

Components and Organization of the Cell

Why motors?: The living cell is organized

Overview: Motors, and where they are important

1. Cytoskeleton, including AFs, MTs, and motors

2. Role of Cytoskeleton: Cargo transport

3. Cell Migration

4. Role of Cytoskeleton: Cell division

5. Communication of Cell with outside world: endocytosis, exocytosis, control of receptors

6. Role of Cytoskeleton: Neuronal Cells

On MTs: kinesin dynein

On Actin:

myosin

General summary:The road systems in the cell

Transport requires motors + roads!

• Microtubules stiff, arranged in a radial fashion

• Actin random and everywhere

• MT Highway system

• Actin Local Roads

self-organization of cytoskeleton, especially interactions with motors (allows feedback)

Regulation

Kinesin Myosin-V Dynein

Head (ATPase)

1

43

5

c6

2

Head(ATPase)

Lever (?)

StalkPi

Pi

KAPP

KHC

KLC

KR2

KR3

Cargo

Ca2+

MR2

MR1

Cargo

KR1

Dynactinbinding

MT binding

Three families of molecular motors

Processivity: porters vs rowers

Processive (porter)

Non-processive (rower)

Images: MCRI Molecular motors group

Kinesin is Processive; Myosin II (muscle) is not. Why?

• A processive motor doesn’t let go of the substrate (MT or AF) so the cargo doesn’t diffuse away

• Many processive motors could get in each others way--all bound to the filament at the same time

• A non-processive motor lets go of the filament at some point in its enzymatic cycle. Thus, multiple motors don’t get in each others way--not active at exactly the same time

• Collective Velocity Different!

Cartoon: kinesin

Nucleotide hydrolysis conformational change AND changes in MT affinity

Monte-Carlo simulation: single motor

Pstep, Pdetach2, and Poff are load dependent

Simulation reproduces known single-molecule kinesin function

We’ll return to theory later to investigate multiple-motor transport

Homework: Simulate a motor walking (Very simple Monte Carlo simulation)

Steps in simulation:

1.Check if bound (bound =true?)

2.If bound=true continue to 3, otherwise end ‘run’, determine how many steps were taken by looking at counter.

3.Check if tries to step (probability check)

if tries to step either

a) falls off (decide which with

b) steps probability check)

4. If steps increment step counter

5. If falls off set bound =false.

Each such set of steps simulates a single walking motor. Repeat many times to get a distribution of ‘runs’

Homework, (hints): Key point in simulation: size of timestep, and probability at each attempt.

Suppose that you know that kinesin takes 8 nm steps, and on average goes 800 nm/sec. That means approx. 100 steps/sec.Can you do a simulation, with time steps of 1/100th of a sec.?(NO!!)

Suppose timestep = 1/10000 sec. What is the probability of stepping in any given timestep?Hint: if 1/10000 sec is the timestep(ts), then 10000 ts/sec, and to get the correct average velocity, 100 of them should turn into actual steps. (P)*10000=100 p = 100/10000=1/100, i.e. at any single time step there is a 1 in 100 chance of stepping, and a 99/100 chance of doing nothing.

Different Theoretical approaches to understand motors

1. Monte-Carlo simulations, e.g based on the simple kinetic approach I just showed (see e.g Kunwar et al, Curr. Biol. 18(16):1173-83 (2008).) (can get from my website: bioweb.bio.uci.edu/sgross)

2. Masters Equations (see e.g Klumpp, Lipowsky, PNAS)

3. Will hear more from Dr. Joanny

Cargo transport

MT based dispersion of pigment

MT based aggregation of pigment

Cell Division: an exciting example of dynamic self-organization

Bio D103 – SS1 2009 lect 20-22 © Grün - all rights reserved

Why?

• Need addtl cells for development

• Need cells to maintain/repair body (+ adaptation).

Requirements for typical cell division:

• Increase cell size (except early embryogenesis).

• copy genome accurately.

• Segregate duplicated genome to each of daughter cell.

• Segregate organelles to each daughter cell.

Incorrect replication/regulation causes problems!!

• Insufficient division growth retardation, degeneration.

• Excessive division birth defects, cancer.

• Incorrect segregation of chromosomes similar effects.

Cell Replication: big picture

25Bio 103-Fall 2009 © Steven Gross lecture 1 © Gross - all rights reserved.


Mitosis & Cytokinesis

Recommended Reading: MBOC 5e, pages 1069 - 109026

lecture 1 © Gross - all rights reserved.


No new protein synthesis after entry into mitosis- better retain proteins required for life immediately after cytokinesis !

Condense chromosomes before trying to separate them- think you can quickly separate many balls of unwound string ?

Disassemble nuclear envelope- can you connect chromosomes to the centrosomes while the nuclear envelope is in between them ?

Ensure that sister chromatids can be reliably and quickly sorted one per daughter cell- how do you assemble a dynamic scaffold that allows us to move chromosomes ?

Ensure that a set of chromosomes and adequate organelles are partitioned to each daughter cell- better make sure that you divide the cell in the right place, and move things appropriately first !

Disassemble the scaffold, reform the nuclear envelope, allow chromatin to reorganize- How to accomplish this without significant new protein synthesis ?

27

Mitosis & cytokinesis - what must the cell accomplish ?

Bio 103-Fall 2009 © Steven Grosslecture 1© Steven Gross


Mitotic microtubules have a reduced half-life due primarily to increased catastrophes

•In mitotic cells, rate of elongation is about twice as fast as that in interphase cells, but shortening occurs at same rate. •Although microtubules in interphase cells undergo catastrophe, they are able to recover.•In contrast, when microtubules undergo catastrophe in mitotic cells, the frequency of recovery is greatly reduced.•Catastrophes also MUCH more common Mitosis

Figure illustrating recovery from catastrophe in interphase cells

Why are there more catastrophes?Why don’t they recover?

28 lecture 1© Steven Gross


Dynamic instability of microtubules is regulated by multiple factors

•Local concentration of dimeric tubulin available for incorporation into growing microtubules.•e.g. a microtubule binding protein, such as stathmin, can reduce the local concentration of free dimeric tubulin. This would increase the likelihood of the GTP cap becoming hydrolyzed and the MT becoming unstable.

•Binding of microtubule associated proteins (MAPs) to microtubules.•Some MAPs can increase the stability of the MT ends, while others (such as kinesin-13, a catastrophin) can decrease the stability.



Three microtubule-based structures are required for mitosis

•Three distinct types of microtubule (MT) structures form during mitosis - astral, interpolar and kinetochore MTs.

•Astral MTs - project from the poles and orient the spindle via interactions with the cell cortex.

•Interpolar MTs - can be free at either end, can penetrate between and through chromosomes, minus ends are close to centrosomes, overlap in zones of inter-digitation.

•Kinetochore MTs - plus ends are embedded in kinetochore, minus ends are at or near spindle pole (aka centrosome), each human kinetochore captures around 20 independent MTs.

30 lecture 1 © Gross - all rights reserved.


•Kinesins 4, and 10 (both + end directed) are chromokinesins that bind to chromosomes and move chromosomes to + end of kinetochore MT.

•Kinesin 13 (not shown; at kinetochore) is a catastrophin that can mediate MT shrinkage.

•Kinesin 5 (+ end motor) contains two motor domains that interact with anti-parallel (inter-polar) MTs, and slide them apart (pink arrows).

•Kinesin 14 (- end motor) contains a single motor domain, but can cross-link MT (i.e. one MT is cargo) and pull them together (blue arrow).

•Dynein / dynactin (- end motor) can bind the cell cortex as cargo, and pull astral MT out from centrosome.

Motor proteins govern spindle assembly and function


Neuronal cell function

Transport by motors: ~ 5 hrs

(Harvard BioVisions)

Diffusion: ~ 7-8 Months D ~ 0.5 x 10-7 cm2/s

Sattelle et al., Eur Biophys J. (1987)

Microtubule-Based Molecular Motors Enable Transport

Particle: 100nm

Distance: 1cm

New materials (eg. neurotransmitter)

Old materials, External survival factors

Transport in Axons: Remarkable and Crucial Task

Axon

MuscleCentralNervousSystem Axon ~ 1m

Cell Body Axon Terminal

Herpes virus particles moving in an axon

2m

ww

w.a

lzh

eim

er-

ne

t.c

h

Specific Challenges in Small-caliber neurons

Cell Body Axon Terminal

Relatively easy to move (viscosity < 10X water)

Harder to move (effective viscosity larger due to no-slip bdry conditions, and difficulty displacing obstacles)

2 recent papers: Theory: J. Wortman et al, Biophys J. (2014) Expt: BR Narayanareddy et al, “Mitochondrial Movement:

Differences Between Transport in Neuronal Cell Bodies Versus Processes” Traffic (2014)

Challenges in understanding motor function (mostly solved):1) how do single molecules work?2) how can we determine the function of molecules too small to see by eye? 3) experimental ways to investigate function of single molecules

Challenges in understanding transport/motor function (not so solved):1) Regulation: how is all of this controlled?2) single-molecule ENSEMBLE 3) Properties of ensembles: how many motors, where in cells, etc.4) how do groups of proteins function together?5) Why choose a specific motor/motors for a specific job—what makes a motor well suited for a particular function?

An introduction to Molecular Motors: from single molecules to function in the cell Steven Gross...

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