Quantitative analysis of changes in actin microfilament contribution to cell plate development in plant cytokinesis.
BACKGROUND
Plant cells divide by the formation of new cross walls, known as cell plates, from the center to periphery of each dividing cell. Formation of the cell plate occurs in the phragmoplast, a complex structure composed of membranes, microtubules (MTs) and actin microfilaments (MFs). Disruption of phragmoplast MTs was previously found to completely inhibit cell plate formation and expansion, indicative of their crucial role in the transport of cell plate membranes and materials. In contrast, disruption of MFs only delays cell plate expansion but does not completely inhibit cell plate formation. Despite such findings, the significance and molecular mechanisms of MTs and MFs remain largely unknown.


RESULTS
Time-sequential changes in MF-distribution were monitored by live imaging of tobacco BY-2 cells stably expressing the GFP-actin binding domain 2 (GFP-ABD2) fusion protein, which vitally co-stained with the endocytic tracer, FM4-64, that labels the cell plate. During cytokinesis, MFs accumulated near the newly-separated daughter nuclei towards the emerging cell plate, and subsequently approached the expanding cell plate edges. Treatment with an actin polymerization inhibitor caused a decrease in the cell plate expansion rate, which was quantified using time-lapse imaging and regression analysis. Our results demonstrated time-sequential changes in the contribution of MFs to cell plate expansion; MF-disruption caused about a 10% decrease in the cell plate expansion rate at the early phase of cytokinesis, but about 25% at the late phase. MF-disruption also caused malformation of the emerging cell plate at the early phase, indicative of MF involvement in early cell plate formation and expansion. The dynamic movement of endosomes around the cell plate was also inhibited by treatment with an actin polymerization inhibitor and a myosin ATPase inhibitor, respectively. Furthermore, time-lapse imaging of the endoplasmic reticulum (ER) revealed that MFs were involved in ER accumulation in the phragmoplast at the late phase.


CONCLUSIONS
By expression of GFP-ABD2 and vital staining with FM4-64, the dynamics of MFs and the cell plate could be followed throughout plant cytokinesis in living cells. Pharmacological treatment and live imaging analysis also allowed us to quantify MF contribution to cell plate expansion during cytokinesis. Our results suggest that MFs play significant roles in cell plate formation and expansion via regulation of endomembrane dynamics.
Higaki
Takumi
T
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha Kashiwa, Chiba 277-8562, Japan. takumi.higaki@gmail.com
Kutsuna
Natsumaro
N
Sano
Toshio
T
Hasezawa
Seiichiro
S
D000199:Actins / Q000502:physiology*; D048429:Cell Size; D002473:Cell Wall / Q000502:physiology*; D002478:Cells, Cultured; D048749:Cytokinesis; D004721:Endoplasmic Reticulum / Q000502:physiology; D011992:Endosomes / Q000502:physiology; D049452:Green Fluorescent Proteins; D006131:Growth Inhibitors / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D018942:Macrolides / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009218:Myosins / Q000502:physiology; D010940:Plant Proteins / Q000502:physiology; D011993:Recombinant Fusion Proteins; D013194:Staining and Labeling; D013997:Time Factors; D014026:Nicotiana / Q000166:cytology* / Q000187:drug effects
D000199:Actins; D006131:Growth Inhibitors; D018942:Macrolides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C104924:bistheonellide A; D049452:Green Fluorescent Proteins; D009218:Myosins