Transcript ********* 1 - Botanik in Bonn

PCDU seminar
14.12.15
Plant-specific Myosins VIII, IX
Fotiadou Liana
Overview
1. Introduction
• The myosin family of motor proteins
• Structure domains of myosins
2. Myosin IX
• Structure
• Myosin XI involvement in cytoplasmic streaming
• Mutual regulation between actin and myosin XI?
3. Myosin VIII
• Structure
• Localization along new walls
• Accumulation in plasmodesmata
• Myosin VIII regulates plasmodesmatal gating?
“-” myosin superfamily
“+”
The
of motor proteins
Hartman, A. The myosin superfamily at a glance, Journal of cell science 2012
Myosin heavy chains
Motor: actin binding, ATP hydrolysis
Coiled-coil: dimerization
Neck: Interacting sites with light
chains , lever arm
Globular tail: Cargo binding
Ca 2+ dependent CaM forms the light chain
2+
Ca conc > 10μM=> CaM dissociation:
decrease of actin-myosin afinity
Shortening of neck domain and step size
Tominaga, Frontiers in Plant Sc., 2012
=> Myosin motility is affected by Ca 2+ concentration and the presence of calmodulin
Overview
2. Myosin IX
• Structure
• Myosin XI involvement in cytoplasmic streaming
• Mutual regulation between actin and myosin XI
2
Myosin XI structure
150-270 kDa predicted MW
Extra N-terminal SH3-like domain before motor region, interactions
with proteins containing SH3 domains.
Neck region with up to 4-6 IQ motifs for CaM binding as light chains.
DIL domain before C-terminal is a signature element for class V and IX myosins.
Cytoplasmic streaming
cell wall
vacuole
cytoplasm
Myosin XI is involved in cytoplasmic streaming
A Chara internode cell can reach up to 10 cm length.
=> Cytoplasmic streaming must reach high velocity for proper mixing of metabolites.
The fastest molecular motor in the world.
The Chara myosin XI can slide the F-actin at
50–60 mm/s.
Subcellular defects linked to Myosin XI function
A. Fluorescent markers expressed in myosin XI knock-out mutants
B. Tail overexpression in cells expressing various organelle markers
Did not reveal a simple 1-1 relationship between myosin isoforms and specific
organelles!
Myosin motors have a direct effect on actin filaments
Luis Vidali et al. Plant Cell 2010;22:1868-1882
Loss of actin axial orientation in P. patens
myosin XI RNA-i cells
Myosins might introduce tension to
filaments, that ultimately leads to their
proper alignment.
=>Mutual regulation of tracks and motors?
Valera V. Peremyslov et al. Plant Cell 2010;22:1883-1897
Skewed actin cables in A. thal 3KO
myosin XI mutants
Overview
3. Myosin VIII
• Structure
• Localization along new walls
• Accumulation in plasmodesmata
• Myosin VIII regulates plasmodesmatal gating?
2
Myosin VIII-consensus structure
120-140 kDa predicted MW
Neck region with up to 3-4 IQ motifs for CaM binding as light chains
Extra N-terminal SH3 and PEST peptide domain before motor region.
PEST cleaved in vitro by protease. Involved in myosin VIII turnover?
Unique C-terminal region, , highly conserved among members
• putative phosphorylation site
• cluster of basic residues
ATM1 : First plant myosin cloned and sequenced
Myosin VIII localizes along cytokinetic plates
G1
Antibody raised against a recombinant tail region
of ATM1
Immunostain
Fluorescence microscopy
• L. sativum, Z. mays, A.thaliana
• Root cells
Myosin localized at peripheral region, underlying transverse cell walls
Longitudinal walls showed punctate labelling
Reichelt et al., 1999
Myosin VIII accumulates at plasmodesmata
Reichelt et al., 1999
Indirect immunogold labelling
Electron microscopy
Labelling detected:
 Along the newly formed cell wall between two cells.
On thin membranous structures going through the cell wall (plasmodesmata).
PD are contractile gates for cell-cell transport
Zambryski and Crawford, 2000
• Plasmodesmatal permeability is regulated by intracellular Ca2+ concentrations (HoldawayClarke et al., 2000)
•Depolymerization of F-actin dilates plasmodesmata (Ding et al. 1996)
•Inhibition of myosin ATPases constricts plasmodesmal necks (Radford and White 1998)
Myosin VIII regulates plasmodesmata gating?
Desmotubule
Actin
Plasma
membrane
František Baluška et al. Plant Physiol. 2001
Take-home message
Myosin XI
• Involved in cytoplasmic streaming and organelle trafficking
• Regulates actin alignment by introducing tension to the filaments
Myosin VIII
• Less important to intracellular motility
• Accumulates at plasmodesmata and sites of new cell wall formation
• Possibly regulates plasmodesmatal gating
References
• Madison S. L. , Nebenführ A., Understanding myosin functions in plants:
are we there yet?, Cur. Op.in Plant Biol., Dec. 2013
• Tominaga, Motoki, and Akihiko Nakano. "Plant-specific myosin XI, a
molecular perspective." Frontiers in plant science, 2012
• Liu B. (ed.), The Plant Cytoskeleton, Ad. in Plant Biol. 2, 2011
• Volkmann, D., et al. "Unconventional myosins of the plant-specific class
VIII: endocytosis, cytokinesis, plasmodesmata/pit-fields, and cell-to-cell
coupling." Cell biology international, 2003
• Baluška, F., et al. Sink plasmodesmata as gateways for phloem unloading.
Myosin VIII and calreticulin as molecular determinants of sink strength?,
Pl. Physiology, 2001
• Reichelt, S,et al. Characterization of the unconventional myosin VIII in
plant cells and its localization at the post-cytokinetic cell wall. The Plant
Journal, 1999
Thanks for your attention!