How cells control the size of their
organelles?
O'Connell and Khodjakov, J Cell Sci 2007
Ishikawa, and Marshall, Nature reviews 2011
Lishibanya Mohapatra
Kondev group
Examples of length regulated cytoskeletal
structures
O'Connell and Khodjakov, J Cell Sci 2007
Mitotic spindle
Examples of length regulated cytoskeletal
structures
O'Connell and Khodjakov, J Cell Sci 2007
Mitotic spindle
Ishikawa, and Marshall, Nature reviews 2011
Cilia
Examples of length regulated cytoskeletal
structures
Size of the structure is maintained despite
a rapid turnover of the components!
O'Connell and Khodjakov, J Cell Sci 2007
Mitotic spindle
Ishikawa, and Marshall, Nature reviews 2011
Cilia
Simple model for the growth of cytoskeletal
structures
Simple model for the growth of cytoskeletal
structures
Simple model for the growth of cytoskeletal
structures
For cytoskeletal structures,
tubulin dimers or actin subunits
Simple model for the growth of cytoskeletal
structures
nm
micron
Simple model for the growth of cytoskeletal
structures
nm
micron
For the structure to have a specific length, either the
assembly or disassembly has to be length dependent
A slight detour..
Length-dependent rates are required
for size- control!
𝛾
r
𝛾
r
Length-dependent rates are required
for size- control!
𝛾
r
r
𝛾
r(l)
r(l)
𝛾(l)
𝛾(l)
Length-dependent rates are required
for size- control!
𝛾
r
r
𝛾
Length-dependent feedback is critical!
r(l)
r(l)
𝛾(l)
𝛾(l)
How can the assembly/disassembly
rates be made length-dependent?
Organelle growth control through limiting
pools of cytoplasmic components
Goehring and Hyman ‘12
Assembly
Disassembly
Assembly depends on the number of free
monomers
Organelle growth control through limiting
pools of cytoplasmic components
Goehring and Hyman ‘12
Assembly
Disassembly
What happens when there are structures
of multiple length scales?
Diversity in actin structures
Cytokinetic actin ring
Filopodia
lammelipodium
sarcomere
Diversity in actin structures
Cytokinetic actin ring
lammelipodium
Different structures coexist in the cell and share a common
pool of monomers. How are their sizes controlled?
Filopodia
sarcomere
Plan of the talk
• Properties and limitations of finite pool
mechanism; the need for additional size control
mechanisms.
• An example of a length control in budding yeast
cells.
• Using size distributions to characterize and
distinguish between multiple size control
mechanisms.
Plan of the talk
• Properties and limitations of finite pool
mechanism; the need for additional size control
mechanisms.
• An example of a length control in budding yeast
cells.
• Using size distributions to characterize and
distinguish between multiple size control
mechanisms.
Limiting monomer pool mechanism
Free monomers
Limiting monomer pool mechanism
Peaked distribution of size
Free monomers
Limiting monomer pool mechanism
A single elongator creates a steady-state filament of a well
defined size.
Limiting monomer pool mechanism
Free monomers
Limiting monomer pool mechanism
Structure 1
Free monomers
Structure 2
Limiting monomer pool mechanism
Competition of different elongators results in a single
‘winning’ structure.
Limiting monomer pool mechanism
Free monomers
Limiting monomer pool mechanism
Structure 1+ Structure 2
Structure 1
Structure 2
Free monomers
Limiting monomer pool mechanism
Two identical elongators yield filaments characterized by
large fluctuations
We need additional
length-control mechanisms to
get structures of distinct lengths!
Plan of the talk
• Properties and limitations of finite pool
mechanism; the need for additional size control
mechanisms.
• An example of length control in budding yeast
cells.
• Using size distributions to characterize and
distinguish between multiple size control
mechanisms.
Actin structures in yeast
~ 5 microns
Budding yeast cell
Actin structures in yeast
Actin Patches
endocytosis
(Arp2/3 complex)
~ 5 microns
Budding yeast cell
Actin Cables
intracellular transport
and polarized growth
(Formins)
Actin cables in yeast
Formins
Wild type
smy1∆
Actin cables in yeast
Formins
Actin cables in yeast
Formins
Wild type
smy1∆
How is the length of the actin
cables controlled by the cell?
Formin(Bnr1) inhibition
by Smy1 using TIRF microscopy
Actin
Actin
Formin
Actin
Formin
Smy1
Formin(Bnr1) inhibition
by Smy1 using TIRF microscopy
Actin
Actin
Formin
Actin
Formin
Smy1
How does Smy1 get to the formins at budneck?
Formin
Formins are localized to the budneck during budding
How does Smy1 get to the formins at budneck?
Formin
Smy1
Smy1’s are everywhere in the cell
How does Smy1 get to the formins at budneck?
Formin
?
Smy1
How does Smy1 get to the formins at budneck?
Formin
•Free diffusion?
?
Smy1
How does Smy1 get to the formins at budneck?
Formin
•Free diffusion?
?
Smy1
•Active transport?
Smy1 is transported by myosin motors on
actin cables
Smy1-GFP
Smy1 is transported by myosin motors on
actin cables
Slower
myosin
Smy1 and myosins are together
Smy1 is transported by myosin motors on
actin cables
Smy1s wait at budneck for a sec
Slower
myosin
Pausing
Smy1 and myosins are together
How does Smy1 get to the formins at budneck?
Formin
•Free diffusion?
?
Smy1
•Active transport?
Antenna mechanism of length regulation
wL
Formin
Smy1
Delivery of Smy1 by myosin motors leads to a length-dependent polymerization rate.
Antenna mechanism of length regulation
wL
Smy1
Formin
Delivery of Smy1 by myosin motors leads to a length-dependent polymerization rate.
koff
wL
Antenna mechanism of length regulation
wL
Smy1
Formin
Delivery of Smy1 by myosin motors leads to a length-dependent polymerization rate.
Assembly
koff
wL
Disassembly
Stochastic model of cable assembly
Rate
Transition
On
Off
Off
On
Model makes predictions for how the distribution of cable lengths
depends on control parameters that can be tuned experimentally.
Effect of Smy1 deletion on cable length
Effect of Smy1 mutation on cable length
Other control mechanisms in cables: Filament
severing?
Silvia Jensen, Goode Lab
Actin filaments
Severing Proteins
Severed actin filaments
Other control mechanisms in cables: Filament
severing?
A longer filament will get severed more!
Other control mechanisms in cables: Filament
severing?
Rates
Assembly
Disassembly
Length
A longer filament will get severed more!
How to distinguish between additional
length-control mechanisms?
Finite pool + Smy1
Severing + Smy1
How to distinguish between additional
length-control mechanisms?
Finite pool + Smy1
Severing + Smy1
What are the predicted length
distributions for weak Smy1
mutants?
How to distinguish between additional
length-control mechanisms?
Probability
Finite pool + Smy1
Length
Width of distribution decreases!
Severing + Smy1
How to distinguish between additional
length-control mechanisms?
Severing + Smy1
Probability
Probability
Finite pool + Smy1
Length
Width of distribution decreases!
Decreasing
Formin-Smy1
binding strength
Length
Width of distribution increases!
Length sensing mechanisms in microtubules
Varga et al. ‘09
Disassembly
Rates
Depolymerisers
Assembly
Length
What do length control mechanisms do?
Modulate assembly rate
Modulate disassembly rate
Disassembly
𝛾
Disassembly
𝛾
Assembly
Assembly
l*
l
• Finite monomer pool
• Dampers (Smy1)
l
l*
• Depolymerisers
• Severing
Plan of the talk
• Properties and limitations of finite pool
mechanism; the need for additional size control
mechanisms.
• An example of length control in budding yeast
cells.
• Using size distributions to characterize and
distinguish between multiple size control
mechanisms.
Glossary of length control mechanisms
Steady state probability distribution
Mean
Glossary of length control mechanisms
Steady state probability distribution
Mean
Using distributions to distinguish
length-control mechanisms
Schematic
Length Distribution
Mean
Finite monomer pool
Variance
Summary
• Finite pool mechanism may not be enough to
make multiple structures; we need additional
size-control mechanisms.
• Smy1 acts as a length sensor in actin cables.
• We can use resulting size distributions to
characterize different size-control
mechanisms.
Thanks!
Thibaut Lagny, MBL
Rob Phillips, Caltech
Goode Lab, Brandeis
Kondev Group
Brandeis
Extra
Future Directions and Applications
Making two structures from a common pool
Future Directions and Applications
Steph Weber, Princeton
Nucleolar size
Future Directions and Applications
Wallace Marshall, UCSF
Nucleolar size
Cilia in Chlamydodmonas
Actin cytoskeleton
Limiting pool mechanism of size control
Goehring and Hyman ‘12
Assembly
?
Disassembly
Additional mechanisms of size control are required to maintain multiple structures
from the same limiting pool.
Glossary of length control mechanisms
Formin(Bnr1) inhibition
by Smy1 using TIRF microscopy
Smy1 is transported by
myosin motors on actin
cables
Actin
Smy1-GFP
Actin
Formin
Actin
Formin
Smy1