Visualizing protein complex movement in plant cell walls

Research Areas:

Developmental biology, Plant biology, Cell biology, Cell wall growth

Imaging Needs:

High resolution and high sensitivity over time in living cells. Visualizing the movement of protein complexes.

Imaging System:
  • Perkin-Elmer Ultraview VoX spinning disk system
  • Leica DM1600 microscope
  • 63X glycerol objective (NA=1.3)
  • Temperature controlled stage and objective lens heater
  • Citrine-YFP excited with 514 nm laser
  • Hamamatsu 9100-02 CCD camera
  • VOLOCITY software
  • Emission band filters 540/30
Imaging Dynamic Systems

Find out how Fang Huang, Jeorg Bewersdorf and colleagues use the sCMOS technology in the ORCA-Flash4.0 camera to achieve video-rate imaging at nanometer scales. Read now.

THE QUESTION

How do plant cells manage cellulose addition and organization during cell growth?

Cellulose microfibrils are an important structural component of vascular plant cell walls, helping to provide tension against turgor pressure. Cellulose is synthesized by multiple cellulose synthetase (CesA) subunits that form a complex—cellulose synthetase complex (CSC)—in the plasma membrane, but how the subunits interact to generate a functional enzyme complex is unclear.

THE BARRIERS

Understanding is hampered by the critical nature of CesA—most previously discovered CESA1 mutants show severe constitutive or conditional phenotypes that fail to produce viable plants.

THE SOLUTION

The anisotropy1 D604N mutation in the Arabidopsis cellulose synthase1 catalytic domain reduces cell wall crystallinity and the velocity of cellulose synthase complexes.
Fujita M, Himmelspach R, Ward J, Whittington A, Hasenbein N, Liu C, Truong TT, Galway ME, Mansfield SD, Hocart CH, Wasteneys GO.
Plant Physiol. 2013 May;162(1):74-85. PMCID: PMC3641231.

Fujita, et al,1 identified a missense mutation in the CESA1 gene of Arabidopsis thaliana that produced viable plants, enabling studies into the role of CESA1 in cell wall growth and morphology. These mutant plants, anisotropy1 (any1), are dwarfs with altered cell morphologies.

Interestingly, the any1 mutation did not alter the amount of cellulose produced, but rather affected the proportion of crystalline cellulose present in the walls. Using a Hamamatsu ImagEM camera to image YFP-labeled CSC movement in wild type and any1 cells, Fujita, et al,1 were able to show that the any1 mutation causes CSCs to move at a significantly slower rate than in wild type cells.

Further studies will be needed to elucidate whether this alteration in CSC movement is a result of impaired catalytic activity or perturbations of protein-protein interactions among CesA subunits or between the CSC and other cellular components.

THE POSSIBILITIES

Fujita, et al,1 used an EM-CCD camera to visualize the movement of YFP-labeled CSCs in living plant cells, collecting images every 10 seconds for 5 minutes. The EM-CCD technology enables sensitive and precise localization, but similar studies can also now be performed using Hamamatsu’s ORCA-Flash4.0 Gen II scientific CMOS camera.

To see how the ORCA-Flash4.0 can be used for video-speed imaging at nanometer scales, read our bench story, Exciting Advances Push the Limits of Visualization, which highlights recent imaging advances from Fang Huang, Jeorg Bewersdorf, and colleagues.

References

  1. Fujita, et al. The anisotropy1 D604N Mutation in the Arabidopsis Cellulose Synthase1 Catalytic Domain Reduces Cell Wall Crystallinity and the Velocity of Cellulose Synthase Complexes. Plant Physiol. 2013 May; 162(1): 74–85. PMCID: PMC3641231
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