Vesicles and actin are targeted to the cleavage furrow via furrow microtubules and the central spindle

Abstract

During cytokinesis, cleavage furrow invagination requires an actomyosin-based contractile ring and addition of new membrane. Little is known about how this actin and membrane traffic to the cleavage furrow. We address this through live analysis of fluorescently tagged vesicles in postcellularized Drosophila melanogaster embryos. We find that during cytokinesis, F-actin and membrane are targeted as a unit to invaginating furrows through formation of F-actin-associated vesicles. F-actin puncta strongly colocalize with endosomal, but not Golgi-derived, vesicles. These vesicles are recruited to the cleavage furrow along the central spindle and a distinct population of microtubules (MTs) in contact with the leading furrow edge (furrow MTs). We find that Rho-specific guanine nucleotide exchange factor mutants, pebble (pbl), severely disrupt this F-actin-associated vesicle transport. These transport defects are a consequence of the pbl mutants' inability to properly form furrow MTs and the central spindle. Transport of F-actin-associated vesicles on furrow MTs and the central spindle is thus an important mechanism by which actin and membrane are delivered to the cleavage furrow.

Figure 1.

Live imaging of cycle 14 mitotic domains. (A) Tubulin-GFP. Dividing cells are clustered in mitotic domains (box). (B) Magnification of box in A. (C) Dlg-GFP. Cells within a mitotic domain enter mitosis as a cluster and divide in a characteristic order. (D) Dlg-GFP is uniform cortical during cytokinesis. Bars: (A) 10 μm; (B–D) 5 μm.

Figure 2.

Vesicles and membrane are targeted to furrows during cytokinesis in cycle 14 cells. All images are from living embryos. (A) Syt-GFP–labeled vesicles. Arrows indicate a single vesicle. Arrowheads indicate other vesicles moving toward the furrow. Time points (s) are shown at the top left of each panel. For the full video, see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200803096/DC1. (B) n-Syb–GFP–labeled vesicles show strong furrow enrichment at cytokinesis (arrowhead). (C) Dah-GFP vesicles show cytoplasmic puncta at the cell equator and furrow enrichment at cytokinesis (arrows). (D) The general membrane dye FM1-43 accumulates at the cell equator during cytokinesis. Arrows, cleavage plane. (E) Grasp-65-GFP–labeled Golgi do not accumulate at the cell equator at cytokinesis. Arrows, cleavage plane. Outline, cell cortex. (F) RhoA-GFP. (G) Percentage of vesicles localized to polar regions of the cell (outer) and to the middle of the cell (center). Cells were divided into thirds (inset). Vesicle localization was scored at cell stages indicated on the x axis. n = 92 (late metaphase), 74 (anaphase B), 96 (furrow initiation), 91 (elongation), or 100 (scission). Bars, 5 μm.

Figure 3.

Actin puncta are targeted to ingressing furrows. (A) Phalloidin in fixed embryos shows F-actin puncta in cycle 14 interphase cells (arrow). (B) F-actin puncta localize to the cell equator near furrow MTs (arrowheads) and the central spindle at cytokinesis (arrows). Merged images of F-actin (phalloidin; red) and MTs (α-Tub; green) are shown in the bottom panels. (C–J) Live series of cycle 14 telophase. Rhodamine-actin, red; Tub-GFP, green. Actin puncta accumulate near the central spindle (D, bracket). Arrowheads in C–F show an individual actin puncta moving alongside an MT bundle at the central spindle. Furrow MTs (G, arrowed bracket) align perpendicular to the cleavage plane. Its midpoint contacts the furrow tip (G, arrowhead). Actin puncta are visible near ends of furrow MTs (I, blue arrowhead) and accumulate at the contact point between the leading furrow edge and furrow MTs (G and I, white arrowheads). Arrow in I shows an actin puncta colocalized with the central spindle. Time points (s) are shown at the top right of each panel. For full video, see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200803096/DC1. Bars, 5 μm.

Figure 4.

3D imaging of actin and vesicles. Markers are shown to the left of panels. 3D perspective is indicated at the bottom right panel corners. (A–H) Phalloidin in fixed embryos. Arrows, arrowheads, and asterisks show individual actin puncta that correspond in each rotation (A–F). (G and H) Boxes show the region considered the central spindle for quantifications. (I) Live series of α-Tub and rhodamine-actin. Time points (s) are indicated at the bottom right of each panel. Images were cropped at the furrow (arrows) and rotated 45° to show xy and z planes. Arrowheads indicate actin puncta colocalized with MT bundle. Dotted bracket indicates central spindle. (J) Genotypes and markers are shown at the top right and cell number (n) is shown in bars. Error = SEM. Center region in the right graph includes the middle one-third of the cell in the xy plane. (K and L) Dah-positive vesicles localize to furrow MTs (arrows) and central spindle MTs (arrowheads). Arrows and arrowheads correspond to same vesicle in each rotation (L and M). Step size = 150 nm, except I (200 nm). Bars: (A and G–L) 3 μm; (B, C, and M) 2 μm; (D–F) 1 μm.

Figure 5.

Live imaging shows that actin puncta associate with vesicles and endosomes. Rhodamine-actin (red) was injected into embryos expressing a distinct GFP fusion protein (green; specified in left column). Puncta was scored as colocalized if two puncta moved in unison over a minimum of two consecutive time points (arrowheads). Time points (s) are indicated at the top right of panel. Percentage of colocalization is shown in the right column. (A–F) Live series shows that actin puncta colocalize with Rab7-GFP–labeled late endosomes (A), Dah-GFP–labeled vesicles (B), Syt-GFP–labeled vesicles (C), and Rab5-GFP–labeled early endosomes (D). Actin puncta did not show considerable colocalization with Grasp65-GFP–labeled Golgi (E) or Clathrin light chain–GFP–labeled membrane (F). Insets in C depict single channels for actin (left) and Syt-GFP (right). Error = SEM. Bar, 5 μm

Figure 6.

pbl embryos have defects in vesicle transport. Markers are indicated on the left. Genotypes are indicated on the right. All panels show live images. (A–F) Actin and Syt-GFP vesicles strongly localize to furrows during cytokinesis in WT (A and D). pbl⁵ embryos fail to localize Syt-GFP vesicles to the cell equator in cells with severe furrowing defects (B) and cells with weak furrowing defects (C). pbl⁵ embryos show weak actin recruitment to the cell equator (E and F). The furthest extent of furrow elongation is shown in B′, C′, E′, and F′. For full videos, see Videos 5, 6, and 7, available at http://www.jcb.org/cgi/content/full/jcb.200803096/DC1. Bars, 5 μm.

Figure 7.

pbl mutants have defects in furrow MT and central spindle organization. All panels show fixed embryos. green, α-tubulin and slight γ-tubulin; red, PH3 and Scribble (for all stains except R [green, Pbl; blue, α-tubulin]). (A–L) WT (A–F) and pbl² (G–L) from cycle 14 metaphase (A and G) through cycle 15 interphase (F and L). (A′–L′) Merged images correspond to A–L. (M) Percentage of normal (+++), weak (++), and severely disrupted (+) spindles at various stages in WT and pbl². Metaphase, n = 46 (WT) and 43 (pbl²); anaphase, n = 32 (WT) and 36 (pbl²); early telophase, n = 28 (WT) and 27(pbl²); late telophase, n = 27 (WT) and 24 (pbl²); and scission, n = 53 (WT) and 42 (pbl²). (N) Some Pbl-deficient cells form weak furrows that complete invagination (arrow) but fail to maintain the central spindle. (O and P) Magnification of equatorial region in WT (O and O′) and pbl² (P) cells. O′ and P were taken from C′ and I′, respectively. Furrow and MT structures are labeled in O. Arrowheads and brackets mark the cleavage plane and central spindle, respectively, in O′ and P. pbl² embryos lack furrow MTs and show a weak central spindle (P). (Q) Percentage of normal (+++), weak (++), and severely disrupted (+) furrow MTs during early telophase in WT and pbl². n = 41 (WT) and 33 (pbl²). (R) Pbl shows punctate localization where furrow MTs contact the leading furrow edge (arrows). Bars, 4 μm. Error = SEM among embryos.

Figure 8.

pbl embryos show defects in central spindle maintenance, furrow ingression, and vesicle transport. Genotypes are indicated on the right. All images show live embryos. Fluorescent α-tubulin was injected at cellularization. (A) Central spindle and furrow MT formation in WT. Bracket, central spindle; arrows, furrow MTs. (B) Mitotic cells in pbl⁵ mutants show midzone MTs but lack furrow MTs and a stable central spindle. (C) pbl⁵ phenotype ranges from cells that lack a central spindle to cells with a strong central spindle. Central spindle severity (left) correlates with defects in furrow invagination (middle) and actin puncta mislocalization (right). n is shown in parenthesis. For full videos, see Videos 8 and 9, available at http://www.jcb.org/cgi/content/full/jcb.200803096/DC1. Bars, 5 μm.