PMC

The Pp1-87B phosphatase down-regulates Moe activation. (A) Protein extracts from S2 cells treated with Calyculin A (Caly. A) or dsRNAs targeting Slik and the catalytic subunits of each Drosophila PP1 and PP2A phosphatases were analyzed by Western blotting using antibodies against Moe (bottom) or specific for T559-phosphorylated Moe (P-Moe; top). The same blot is shown at two different expositions (exp.) for P-Moe. flw, Flapwing; mts, Microtubule star. (B) Immunodetection of P-Moe in control S2 cells or after depletion of Slik or Pp1-87B. (C) Dynamics of Moe-GFP during the cell cycle after depletion of Pp1-87B. Arrowheads show persistence of the association of Moe-GFP with the polar cortex. (D) Distribution of GFP–Pp1-87B (top) and Slik-GFP (bottom). Division stages were identified with α-Tubulin-mCherry (not depicted). Bars, 10 µm.

Moe controls polar relaxation and retraction of short-lived blebs. (A) Rapid time-lapse imaging of a cell line coexpressing Moe-GFP and α-Tubulin-mCherry in control condition (top) and after Slik depletion (bottom). The merge image shows maximum projection and close-up single focal planes from the boxed region; Moe-GFP is shown in black. Arrowhead shows an apparent cortical rupture at the site of bleb formation, and arrows point to unregulated blebs. (B) Individual blebs were plotted according to their maximal length in controls cells and after Slik or Moe dsRNA depletion. Bars represent mean values for each condition. (C) Rapid time-lapse imaging of a GFP-Utrophin-CH (GFP-UtrCH; F-actin probe) and α-Tubulin-mCherry cell line in control conditions (top) and after Moe or Slik depletion. Close-ups correspond to the framed region and show the GFP signal in black. Arrowheads show cortical actin rupture and F-actin defective recruitment to the bleb membrane. Bars, 10 µm.

Depletion of Skittles and Pten destabilizes the mitotic cortex. (A) Schematic representation of phosphoinositide (PI) pathways. Each of 31 enzymes was individually inactivated by dsRNA, and its influence on cortical stability was analyzed by time-lapse imaging of α-Tubulin-GFP cells undergoing mitosis (n > 100 cells per condition). (B) The graph plots the percentage of cells with mitotic blebbing in control condition and after inhibition of CG10260, Pten, or Skittles. (C) Cellular levels of PI(4,5)P₂ in controls and in cells after depletion of CG10260, Pten, or Skittles, as measured by biochemical assays. An arbitrary value of 1 was attributed to levels observed in wild-type cells. (D) Influence of Pten and Skittles on PI(4,5)P₂ levels at the cortex of S2 cells. Pictures show GFP-Tubby cells in control conditions and after Pten or Skittles depletion. The graph plots the percentage of cells showing membrane association of GFP-Tubby in each condition. Error bars represent SD. Bars, 10 µm.

Localized activity of Skittles and Pten controls sites of PI(4,5)P₂ production and Moe activation. (A) Dynamic of Pten-GFP distribution during mitosis (top). A stable cell line that expresses Grp1-PH-GFP was used to probe PI(3,4,5)P₃ distribution during mitosis in control conditions or after Pten depletion. (B, top) Time-lapse frames of a Skittles-GFP cell progressing through mitosis. Bottom images show a living cell stably expressing GFP-Tubby after Skittles depletion. (C) FACS analysis showing the distribution P-Moe intensity in mitosis (assessed by histone H3 phosphorylation) of control and Pten- or Skittles-depleted cells. a.u., arbitrary unit. (D) Distribution of Moe-GFP after Skittles depletion. (E) Rapid time-lapse imaging of a GFP-Utrophin-CH (GFP-UtrCH) and α-Tubulin-mCherry after depletion of Skittles showing maximum projection. Close-ups (focal planes) correspond to the framed region and show GFP signal in black. Arrowheads show cortical rupture and defects of F-actin recruitment at the bleb rim; arrows show unregulated mitotic blebs. Bars, 10 µm.

PI(4,5)P₂ regulates Moe distribution during mitosis. (A) Time-lapse frames of cells stably expressing the N-terminal FERM domain of Moe fused to GFP (top) or a full-length mutant form of Moe (Moe-KN-GFP; bottom) at which point mutations in the FERM domain abolish binding to PI(4,5)P₂ (). (B) Dynamics of GFP-Tubby, a PI(4,5)P₂ probe in a living S2 cell undergoing mitosis. Max Proj., maximum projection. (C) Quantification of the equatorial enrichment of GFP-Tubby (green) and of anaphase cell elongation (blue). L, length along the spindle; I, length along the equator. (D) Schematic representation of rapamycin-induced dephosphorylation of PI(4,5)P₂. Protein domains expressed from RC constructs heterodimerize upon rapamycin addition causing dephosphorylation of PI(4,5)P₂ at the plasma membrane (). (E) Cell lines stably expressing GFP-Tubby, Moe-GFP, or Moe-TD-GFP were transfected (+RC) or not transfected (−RC) by RCs. Living interphase or metaphase cells were imaged just before and after rapamycin addition. (F) Rapamycin-treated Moe-GFP cells in telophase expressing (+RC) or not expressing (−RC) RC constructs. Bars, 10 µm.

Model of the spatiotemporal regulation of Moe activity throughout the successive steps of the cell cycle. The cell cortex that comprises the plasma membrane and the underlying cytoskeleton works as a single functional unit to control cell morphology. (A) Two pathways temporally and spatially control coupling of actin filaments to the plasma membrane through the regulation of Moe activity. (B) Redistribution of the Pp1-87B phosphatase at mitosis entry is likely to promote a burst of Slik-dependent phosphorylation of Moe at early mitotic stages and then ensures Moe down-regulation at the end of division and in interphase. The localized enrichment of Skittles and Pten acts to increase PI(4,5)P₂ levels at the anaphase equator, further promoting Moe recruitment at the equator and release at the polar cortex to allow cell elongation. The same players are involved in the transient recruitment and activation of Moe at the cortex of polar blebs, which is important for their efficient retraction. P, phosphorylation.

Control of Moe activation contributes to cell elongation and cytokinesis. (A) Time lapse of Moe distribution throughout the cell cycle observed in a stable S2 cell line coexpressing Moe-GFP (white) and α-Tubulin-mCherry (not depicted). (B) Dynamics of Moe-GFP during anaphase cell elongation. Top images show a single focal plane of Moe-GFP, and bottom images show maximum projections of Moe-GFP and α-Tubulin-mCherry. (C) Quantification of the equatorial enrichment of Moe-GFP (green) during anaphase elongation (blue) measured as the ratio between cell length along the spindle (L) versus the equator (I). (D) Dynamics of Moe-TD-GFP as observed in living cells upon depletion of endogenous Moe. Moe-TD-GFP remained associated with the polar cortex in anaphase (arrowheads) and caused binucleation (asterisks point to nuclei). (E) Distribution of the maximal elongation of individual Moe-GFP or Moe-TD-GFP cells (cell length in anaphase B/cell length in metaphase). Black dots represent cells that successfully passed through division, and red dots indicate cytokinesis failure. Bars represent mean values for each condition. (F) F-actin distribution in cells expressing Moe-GFP or Moe-GFP-TD upon depletion of endogenous Moe. The right-most images show maximum projection (Max. Proj.). (G and H) Polar enrichment of Moe (G) and F-actin (H) in cells expressing Moe-GFP or Moe-TD-GFP in the absence of endogenous Moe. Boxes show top and bottom quartiles, horizontal lines show median values, and vertical lines show minimal and maximal values. Bars, 10 µm.