3/24/14&
1&
Elizabeth Haswell Department of Biology
Washington University in St. Louis
MscS-Like Mechanosensitive Ion Channels: Answering the Osmotic Challenges of Plant Development
Plant Biology
Mechano-biology
Signal Transduction
Plant Mechanotransduction
3/24/14&
2&
Plants are a Well-Established Model System for the Study of Mechanotransduction
The Power of Movement in Plants (1880) Darwin & Darwin
Mechanical Stimuli Osmotic Pressure Turgor Pressure
Gravity Touch
Shear Force
Responses Altered plant morphology Different growth pattern or
direction Organ movement
Altered gene expression Rapid changes in organelle
positioning Increased intracellular [Ca2+] Changes in cytoplasmic pH
Why study the blue arrow? • To answer a fundamental question in biology. • To provide additional examples for comparison with other systems. • To provide a foundation for future agricultural and ecological
improvements.
Mechanotransduction Influences Post-Embryonic Development
Image from Wikimedia; Reviewed in Monshausen & Gilroy (2009) Trends Cell Biol 2009 19:228-35
3/24/14&
3&
Mechanotransduction
appearance of a signal
signal perception by
a receptor
-P
change in cellular state
= the process by which a physical stimulus is transduced into a biochemical signal capable of eliciting a cellular response.
Mechanoreceptor = a protein or cellular structure that
changes activity with applied force.
FORCE
FORC
E
protein is phosphorylated
gene expression is induced
• Can be opened through tension in the membrane.
• When open, allow ions to flow down a concentration gradient.
• Proposed to serve as sensors of gravity, touch, sound, and osmotic pressure in animals, plants, and bacteria.
Mechanosensitive (MS) Ion Channels: Molecular Mechanism for Perception of Mechanical Stimuli
Image adapted from Wilson, Maksaev & Haswell (2013) Biochemistry 5:5708-5722.
tension
membrane
3/24/14&
4&
History: Mechanosensitive Channels in Plants
(2003 - ) Arabidopsis molecular genetics: Identification of candidate mechanosensitive channel-encoding genes. Three families of likely MS proteins identified based on 1) homology to a bacterial channel, 2) homology to a mammalian channel, and 3) on a functional assay.
Pivetti et al. (2003), Nakagawa et al. (2007), Coste et al. (2010).
(1938 - ) Physiology and electrophysiology: Identification of action potentials in giant algal cells. Used as a major model system for the study of electrical signaling, alongside squid axons.
Cole &Curtis, (1938), Cole & Curtis, (1939), Wayne (1994), Shimmen (1997), Kaneko et al. (2009).
L.C. Falke et al. (1988), J.I. Schroeder & R. Hedrich (1989), J. Alexandre & J.-P. Lassalles (1991), D.J. Cosgrove & R. Hedrich (1991), P.-M. Badot et al. (1992), E.P. Spalding & M.H.M. Goldsmith (1993), J.P. Ding & B.G. Pickard (1993a, 1993b), B.G. Pickard & J.P. Ding (1993), A. Garrill, et al. (1994), N. Moran et al. (1996), K. Liu & S. Luan (1998), B.D. Lewis & E.P. Spalding (1998), K. Yoshimura (1999), M. Heidecker et al. (1999), Z. Qi et al. (2004), R. Dutta & K.R. Robinson (2004), Haswell, Peyronnet et al., (2008), Zhang & Fan (2009).
(1988 - ) Patch-clamp electrophysiology: Identification of mechanosensitive ion channel activities. Over 18 separate activities previously identified in a variety of plants, membranes, and cell types:
E. coli Mechanosensitive Channel of Small Conductance (MscS):
Structural data from Bass, Strop, Barclay & Rees (2002) Science 298:1582-7e, 2002 and Lai, Poon, Kaiser & Rees (2013) Protein Sci 22:502-9
Closed state
Image adapted from Wilson, Maksaev & Haswell (2013) Biochemistry 5:5708-5722.
Trans-membrane
domain
Cyto-plasmic domain
• A homoheptamer with 3 transmembrane passes per subunit and a large cytoplasmic domain resembling a Japanese lantern.
• Membrane tension causes the reorientation of transmembrane helices.
• Ions flow into the side portals on the vestibule and out through the pore
• Many structural, biophysical, and functional studies over the past 30 years.
Open state
3/24/14&
5&
0.5M NaCl Osmotic
adjustment
;o)
Martinac, Buechner, Delcour, Adler & Kung (1987) PNAS 84: 2297-301; Levina, Totemeyer, Stokes, Louis, Jones & Booth (1999) EMBO J 18:1730-7; Sukharev (2002) Biophysical J 83:290-8.
MscS Serves as a Bacterial Osmotic Safety Valve
• MscS is a largely non-selective ion channel with a HUGE conductance (~ 1 nS).
Distilled water High osmotic
pressure increases membrane
tension.
MS channels open.
Solutes and water exit, osmotic pressure
is relieved.
;o)
• Along with MscL is required for survival of extreme hypoosmotic shock, such as a shift from 0.5 M NaCl to distilled water.
Conserved?
Two Classes of MscS Homologs are Found in Plant Genomes
C. reinhardtii P. patens A. thaliana O. sativa P. trichocarpa S. pombe
Class I 4 11 3 3 5 0
Class II 3 5 7 3 14 2
Total 7 16 10 6 19 2
Class I proteins are predicted to localize to endosymbiotic organelles.
Class II proteins are predicted to localize to the plasma membrane.
All images from Wikipedia. Adapted and updated from Haswell (2007) Current Topics in Membranes 58:329-53
3/24/14&
6&
= conserved domain
= transmembrane domain
MscS Family Members from Bacteria and Plants Have Diverse Monomer Topologies
= MSL8-specific sequence
Class I Class II
= MSL2-specific sequence
Are these even MS channels?
MSL10-GFP single channel patch clamp
Haswell & Monshausen (2013) J Ex Bot, 64:4663-80; Maksaev & Haswell (2012) PNAS 109:19015-20.
MSL10-GFP Xenopus oocyte 5 days
after RNA injection.
Heterologous expression in Xenopus
oocytes
Patch-clamp Electrophysiology in Xenopus oocytes Demonstrates that Arabidopsis MSLs are MS Ion Channels
Why do plants need MSLs?
3/24/14&
7&
• “ . . . physiological functions are speculative” Nakagawa et al., (2006) PNAS 104:3639-3644.
• “ . . . the physiological function of these proteins also remains enigmatic” Monshausen & Gilroy (2009) Trends Cell Biol 2009 19:228-35.
• “The physiological function . . . however, has not been identified“ Arnadottir and Chalfie (2010) Annual Rev Biophysics 39:111-137.
msl mutant plant
Hyper-osmotic shock Hypo-osmotic shock
Environmental mechanical stresses of
every kind
+ Wild-type response
=
MS Channels Protecting Membranes from
Osmotic Stress
1. Environmental (E. coli MscS)
2. Developmental (A. thaliana MSL8)
3. Intracellular (A. thaliana MSL2/3) —with developmental effects
2. Developmental (A. thaliana MSL8)
3/24/14&
8&
Fertilization in Arabidopsis thaliana
Arabidopsis flower
Why Would Pollen Need MS Ion Channels?
Why Would Pollen Need MS Ion Channels?
Images from Jürgen Berger / Heiko Schoof, Electron Microscopy Unit, Max Planck Institut; Hiscock & Allen, New Phytologist (2008) 179: 286–317.
Stigma cells = part of female gametophyte
Pollen grain = male gameteophyte
Pollen-Stigma Interactions During Pollination
H2O
Ovules = female gamete
3/24/14&
9&
Firon, Nepi & Pacini (2012) Ann Bot 109:1201-1214
Water content <15%
Pollen Desiccation and Re-Hydration: Developmental Osmotic Shock Water
content >50%
Water content
Pollen grain volume
MODEL: MSL8 Serves to Protect the Pollen Grain from the
Osmotic Stresses of Rehydration
MSL8 plays a role in the pollen plasma membrane similar to that played by MscS in the E. coli inner membrane, except that it is protects the cell from
developmental rather than environmental osmotic challenge.
3/24/14&
10&
WHAT’S NEXT?
• Subcellular localization of MSL8 and in situ electrophysiology.
• Better evidence for our model, linking the mutant phenotypes with defects in ion efflux during hydration.
• Evolutionary history of MSL8-type genes; specific to flowering plants?
• Live imaging of pollen tube growth
MS Channels Protecting Membranes from
Osmotic Stress
1. Environmental (E. coli MscS)
2. Developmental (A. thaliana MSL8)
3. Intracellular (A. thaliana MSL2) —with developmental effects
3/24/14&
11&
= conserved domain, pore-lining helix
= transmembrane domain
MscS Family Members from Bacteria and Plants Have Diverse Monomer Topologies
= MSL8-specific sequence
Class I Class II
= MSL2-specific sequence
✔
Plastids From Plants Lacking MSL2 and MSL3 are Round and Enlarged
Leaf epidermal plastids
Plants
expressing plastid-targeted dsRED
Are they under osmotic stress?
3/24/14&
12&
Increasing Cytoplasmic Osmolarity Suppresses the Plastid Size and Shape Defects in the msl2
msl3 Mutant
Veley, Marshburn, Clure & Haswell. (2012). Current Biology 22:408-413.
Plants expressing plastid-targeted dsRED . .
.
. . . . . . .
msl2 msl3
P P
P
P
Veley, Marshburn, Clure & Haswell. (2012). Current Biology 22:408-413.
MS Channels in the Envelope are Required to Relieve Plastid Hypoosmotic Stress of the Cytoplasm
P
P P
P
P
WT
Low water potential High osmolarity
High water potential Low osmolarity
Direction of water flow
• Growth on high osmotic medium. • Genetic lesion. • Withholding water. • Treating excised leaves with
hypoosmotic solution.
P
P P
P
P
Plastids shrink and
regain shape
P P
P
P
Increase cytoplasmic osmolarity
3/24/14&
13&
Working Model: Plastidic MSLs Serve to Protect Plastids from the Osmotic Stresses of the Cytoplasm
MSL2 and MSL3 play a role in the inner chloroplast envelope similar to that played by MscS in the E. coli inner membrane, except that they are required
constitutively to relieve intracellular osmotic stress.
How Do Cells Respond to Osmotically Stressed Plastids?
Arabidopsis mutants lacking mechanosensitive channels in the plastid envelope serve as a sensitized background to study the cellular and developmental response to plastid
osmotic stress.
3/24/14&
14&
Environmental water stress across the plasma membrane: • High salt • Dehydration • Freezing
Increased production of cytoplasmic
osmolytes
Osmotic adjustment
Pro
Pro
Pro Pro
Pro
Pro
In plants, major compatible solute is the amino acid Proline
Pro
Drought Stress Signaling Leads to Osmotic Adjustment
X
Drought stress signaling
Increased accumulation
of ABA ABA
Plant stress hormone
abscisic acid
Osmotically
stressed plastid
?
?
msl2 msl3 Mutants Have Elevated Levels of Proline and ABA
0
5
10
15
20
25
30
35
40
msl2-3 msl2-3msl3-1
msl2-3msl3-1+MSL2g
AB
A Le
vels
(ng/
g of
FW
)
Col-0
*A
Wilson, Basu, Bhaskara, Verslues & Haswell, provisionally accepted.
3/24/14&
15&
0
5
10
15
20
25
30
35
40
msl2-3 msl2-3 msl3-1
Prol
ine
(ȝm
ol g
-1 F
W) MS
MS + Fluridone MS + Fluridone + ABA MS + ABA
Col-0
0
2
4
6
8
10
12
14
16
aba2-1 msl2-3msl3-1
aba2-1msl2-3msl3-1
Prol
ine
(ʅm
ol g
FW
-1)
Col-0
A
B
ABA Biosynthesis is Required for the Pro Accumulation in msl2 msl3 Mutant Plants
Wilson, Basu, Bhaskara, Verslues & Haswell, provisionally accepted.
Thus, msl2 msl3 mutants constitutively exhibit three hallmarks of osmotic stress response: 1) Accumulation of solutes, especially
proline.
2) Elevated levels of ABA.
3) Potentiated ABA responsiveness.
WHAT’S NEXT?
Find the plastid osmosensor and components of the
signaling pathway from the plastid to the water stress
pathway.
Investigate the developmental defects that result from plastid osmotic
stress.
Working Model: Plastids Serve as Osmotic Sensors From Within the Cytoplasm
Increased production of cytoplasmic osmolytes
Osmotic adjustment
Drought stress signaling
Increased accumulation
of ABA X
Osmotically
stressed plastid
Pro
Pro
Pro Pro
Pro
Pro
3/24/14&
16&
Are all MSLs Simple Osmotic Safety Valves?
= conserved domain, pore-lining helix
= transmembrane domain
MscS Family Members from Bacteria and Plants Have Diverse Monomer Topologies
= MSL8-specific sequence
Class I Class II
= MSL2-specific sequence
✔ ✔
3/24/14&
17&
Take Home Messages
MscS-Like channels serve to protect cells from osmotic stresses that are environmental, developmental, and intracellular. msl mutants are a useful tool to study how plants respond to organellar osmotic stress. There is much more to be learned about these channels, about osmotic stress in development, and about mechanotransduction in development!
Team Haswell Postdoctoral Fellows Dr. Grigory Maksaev
Dr. Kira Veley Dr. Maggie Wilson
Graduate Students Eric Hamilton
Angela Schelgel
Technicians Emma January Gregory Jensen
Kelsey Kropp
Undergraduates Meera Basu
Sarah Kloepper Josephine Lee
Samantha Embrick
Ray
Andrew
Anu
Katherine Ellen Kelly Silvano Michael Sarah
Cara
Maia
Meera
Samantha
Greg, Maggie, Grigory Eric, Emma, Sarah Liz, Kira, Kelsey
JOIN US-- POSTDOC POSITIONS AVAILABLE!!
3/24/14&
18&
Doug Rees, Caltech Rob Phillips, Caltech
Daniel Schachtman, Monsanto
Edgar Spalding, U Wisconsin
Adam Cohen, Harvard
Jeff Harper, U Nevada
Paul Verslues, Academia Sinica
Olivier Hamant, University of Leon Darron Luesse, SIUE
Heather Marella, Bridgewater State Chris Wolverton, Ohio Wesleyan
Collaborators and Funding
NSF MCB-0816627
NSF CAREER MCB-1253103
NIH R01GM084211 NIH 2R01GM084211
NASA NNX13AM55G
Gordon and Betty Moore
Foundation
Monsanto
I-CARES
