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First published online 20 July 2004
doi: 10.1242/jcs.01236

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Continuous phosphatidylinositol metabolism is required for cleavage of crane fly spermatocytes

¹ Department of Biology, York University, 4700 Keele Street, Toronto, Ontario, M3J 1P3, Canada
² Program in Developmental Biology, Room 9145 Elm Wing, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada
³ Department of Medical and Molecular Genetics, 1 King's College Circle, University of Toronto, Toronto, Ontario, M5S 1A8, Canada

View larger version (19K): [in a new window]   Fig. 1. Simplified version of the phosphatidylinositol cycle. Inositol is obtained from nutritional sources or de novo synthesis (not shown) or it is recovered from inositol trisphosphate [Ins(1,4,5)P3] by a salvage pathway that involves sequential dephosphorylation of inositol phosphates by the enzymes inositol polyphosphate phosphatase (IPPase; indicated as step 1) and inositol monophosphate phosphatase (IMPase; step 2), both of which are inhibited by lithium. Phosphatidylinositol (PtdIns) is synthesized from inositol and CDP-diacyglycerol (CDP-DAG). Phosphatidylinositol is monophosphorylated to form PtdIns(4)P by type III PtdIns 4-kinase (step 3), which is inhibited by wortmannin (WT) and LY294002 (LY). PtdIns(4)P is phosphorylated by PtdIns(4)P 5-kinase to form phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. This in turn, is hydrolyzed by phospholipase C (PLC; step 4) to form Ins(1,4,5)P3 and DAG in a reaction inhibited by U73122. DAG is then converted to phosphatidic acid (PA) and then CDP-DAG to complete the cycle. Note that WT and LY also inhibit PtdIns 3-kinase (not shown) and that a second type of PtdIns 4-kinase, the type II enzyme, is not inhibited by WT or LY.

View larger version (38K): [in a new window]   Fig. 2. Cytokinesis in control cells proceeds by an exponential or logarithmic decrease in the cleavage furrow diameter. (A) Phase micrographs of a time-lapse series showing cytokinesis of a single primary crane fly (Nephrotoma suturalis) spermatocyte. Pictures were taken at (h:min:s): 15:46:45, 15:49:40, 15:51:47, 15:54:27, 15:58:10 and 16:01:24. Bar, 10 µm. (B) Graph of cleavage furrow diameter of control cell shown in A measured as a function of time. The dashed line represents the computer-generated exponential best-fit curve to the points starting from the second minute.

View larger version (14K): [in a new window]   Fig. 3. Lithium dramatically slows the progression of cytokinesis and this effect is reversible by myo-inositol. Graphs of crane fly spermatocyte cell diameter measured at the cleavage furrow plotted on a logarithmic scale as a function of time for single cells treated with Li+. (A) The effects of 100% Li+ (added at the left arrow) and reversal with Ringer's solution (right arrow). The line on the left is the computer-generated best linear fit to the points between 2 and 4 minutes (i.e. expected curve in the absence of Li+). The dashed line on the right is the computer-generated best linear fit to the included points. (B) The effects of 25% Li+ Ringer's (added at the left arrow) and reversal with myo-inositol added to the Li+ Ringer's (right arrow) for the series of points indicated by +; the dashed line is the best linear fit to the points after addition of myo-inositol. The circles and the other best-fit line are from a control cell. (C) A control cell maintained in Ringer's solution. The line is the best linear fit to the circled points. (D) A cell treated with Li+, then with myo-inositol in Li+, at the times indicated on the graph. Cleavage slowed a few minutes after adding Li+ and sped up after myo-inositol was added. The line is the computer-generated best fit to the points after addition of myo-inositol.

View larger version (20K): [in a new window]   Fig. 4. The average slope of log10 diameter as a function of time for Li+-treated crane fly spermatocytes is dramatically reduced compared to that of control cells and recovers substantially upon myo-inositol addition. The average slopes (±s.d.) are control 0.0647±0.0295 (n=12), Li+ 0.0137±0.0065 (n=12) and Li+ plus myo-inositol 0.0341±0.0140 (n=7). Error bars represent s.d. Values are significantly different for control versus Li+ or Li+ versus myo-inositol (P<0.0004, Student's t-test), whereas the difference between the control and myo-inositol was borderline significant (P=0.021, Student's t-test).

View larger version (14K): [in a new window]   Fig. 5. LY294002 (LY) and wortmannin (WT) block further cytokinesis progression in crane fly spermatocytes. (A,B) Graphs of cleavage furrow diameter as a function of time for single cells treated with either (A) LY (added at the arrow) or (B) WT (added at the arrow). Cleavage furrow diameters in control cells (O) are shown for comparison.

View larger version (24K): [in a new window]   Fig. 6. LY294002 (LY) and wortmannin (WT) affect the extent of cleavage of dividing crane fly spermatocyte cells and the effect of WT is reversible. Bars with dots, average maximum diameters in µm (at the start of cleavage). Clear bars, average minimum diameter at the end of cleavage in control cells (n=8) and at the end of treatment in cells treated with LY (n=7) or WT (n=8), as indicated. Dark gray bars, average diameter after washing out WT (n=4). After washout of WT cytokinesis resumes and the diameters become smaller. Error bars represent s.d. Minimum diameters of control cells were not significantly different from those of washed out WT-treated cells (P=0.3, Student's t-test).

View larger version (103K): [in a new window]   Fig. 7. Phase micrographs of a time-lapse series showing cytoplasmic blebbing in a single crane fly primary spermatocyte after treatment with wortmannin. Blebbing (arrows) is observed at the poles of the cell. The illustrations are at (h:min:s): 17:10:37, 17:13:15, 17:14:47, 17:14:57, 17:15:44 and 17:18:02. Bar, 10 µm.

View larger version (22K): [in a new window]   Fig. 8. U73122 (U7) causes reversible regression of cleavage furrows in crane fly primary spermatocytes. (A) Concentration dependence of U7 in blocking constriction. Clear bars, cells in which there was no effect on cleavage. Hatched bars, cells in which cytokinesis was blocked. (B) Graph of a single cell treated with 4 µM U7 (added at the left arrow), which was then washed out with Ringer's solution (right arrow). Note that after addition of U7, the furrow slows, regresses, and then resumes constriction approximately 8 minutes after washout. The dashed line is the computer-generated exponential best-fit curve through the points prior to addition of U7.

View larger version (105K): [in a new window]   Fig. 9. Treatment with U73122 (U7) results in depletion of cleavage furrow F-actin. Confocal fluorescence micrographs of crane fly primary spermatocytes stained with Alexa-488 phalloidin to visualize F-actin. Each illustration is of a summed Z-series of confocal images. (A,B,C) Control cells in (A) late anaphase, (B) later anaphase and (C) telophase. (D) Cell treated with Li+. (E) Cell treated with LY. (F,G) Cells treated with U7. Actin filaments appear normal in all cells except for those treated with U7, in which patchy cortical actin is prominent at the poles and actin filaments are absent or greatly reduced at the equator (F,G). Bar, 10 µm.

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