(A) Images of fixed WT, , and embryos are shown in cycle-13 metaphase and cellularization (staged by DNA, blue) at the sections crossing the furrow

(B) Compared with WT, RhoGEF2 is more diffuse at the furrow in and embryos at cycle-13 prophase. Green, F-actin; red, RhoGEF2. (C) Line plots of RhoGEF2 (red curves) and Dlg (green curves) signal intensities (normalized to the maximum signal of each plot into a 0–1 scale) crossing the furrows (red lines in B). WT exhibited a single focused RhoGEF2 peak corresponding to the Dlg peak. In and embryos, RhoGEF2 showed unfocused peaks that did not generally correspond with the Dlg peak. (D and E) Rho1 and Diaphanous (red) are not concentrated in furrow regions lacking F-actin (green) but localize properly at regions maintaining F-actin in embryos. (F) At cycle-13 interphase, RhoGEF2 (red) is diffusely localized around the furrow, whereas Anillin (green) is localized normally (arrows). (G) Nuf shows dominant genetic interactions with components of the RhoGEF2–Rho1 pathway. Cycle-13 prometaphase or metaphase embryos (staged by DNA; blue) derived from females with different genetic background were stained with phalloidin to show the furrow structure. (), (), (), (), and () embryos display abnormal furrow morphologies (arrows indicate furrow breaks or weak furrows), whereas () embryos have relatively normal furrow morphology. Summary of phenotypic frequencies is shown in . Bars: (A and G) 10 μm; (B–F) 5 μm.<p><b>Copyright information:</b></p><p>Taken from "Nuf, a Rab11 effector, maintains cytokinetic furrow integrity by promoting local actin polymerization"</p><p></p><p>The Journal of Cell Biology 2008;182(2):301-313.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483530.</p><p></p

(A) In untreated cycle-12 embryos, membrane (GFP-Dlg, green) is closely surrounded by F-actin (red) at the furrow

(B) 2 mM LatA or DMSO was injected at cycle-13 early prophase. GFP-Dlg–marked plasma membrane was progressively lost (arrowheads) as furrows invaginated in LatA-injected embryos (top row). Membrane loss started after most of the F-actin was lost at the furrow (middle row). (C) Furrow integrity was monitored relative to spindle spacing and dynamics (tubulin, red). Spindle fusion (asterisks) occurred where membrane (Dlg, green) was lost at the furrow. See Video 2 (available at ). Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Nuf, a Rab11 effector, maintains cytokinetic furrow integrity by promoting local actin polymerization"</p><p></p><p>The Journal of Cell Biology 2008;182(2):301-313.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483530.</p><p></p

In WT embryos, Nuf promotes actin polymerization at the invaginating furrow by properly localizing RhoGEF2 to the furrow, where it activates Rho1

Up-regulating actin polymerization stabilizes furrow membrane. In mutant embryos, failed RhoGEF2 recruitment results in insufficient actin polymerization. This in turn results in loss of furrow membrane integrity.<p><b>Copyright information:</b></p><p>Taken from "Nuf, a Rab11 effector, maintains cytokinetic furrow integrity by promoting local actin polymerization"</p><p></p><p>The Journal of Cell Biology 2008;182(2):301-313.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483530.</p><p></p

(A) 1 mM Jasp, 2 mM LatA, or DMSO control were injected into GFP-Dlg embryos at cycle-13 early prophase (when furrow is ∼3 μm in length), and furrow invagination was followed over time at depths from −2 μm to −8 μm below the embryo surface

(B) The lengths of invaginating furrows (y axis) over time (x axis) after 1 mM Jasp or DMSO injections in A. (C) Quantification of results in A. Increases in furrow lengths (in micrometers) 8 min after Jasp ( = 5 embryos) or DMSO ( = 5 embryos) injections were analyzed. ***, P < 0.001. Error bars represent SEM. Bar, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Nuf, a Rab11 effector, maintains cytokinetic furrow integrity by promoting local actin polymerization"</p><p></p><p>The Journal of Cell Biology 2008;182(2):301-313.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483530.</p><p></p

(A and B) 1 mg/ml C3 transferase was injected at cycle-13 early prophase

(A) GFP-Dlg–marked furrow membrane (green) was progressively lost and eventually led to spindle fusions (red, asterisks). See Video 8 (available at ). (B) 2 min after C3 injection, most of the F-actin has been lost from the furrow (bottom). In contrast, membrane loss has just started (top). (C) 1 mg/ml RhoA(Q63L) was injected into embryos at cycle-13 early interphase. Green, GFP-Moesin; red, tubulin. At metaphase (18:00), normal furrow structures were maintained in the region close to injection site (arrow); however, loss of F-actin at the furrows is evident further away from the site of injection (arrowheads). See Video 9 (available at ). Bars: (A) 10 μm; (B) 5 μm; (C) 20 μm.<p><b>Copyright information:</b></p><p>Taken from "Nuf, a Rab11 effector, maintains cytokinetic furrow integrity by promoting local actin polymerization"</p><p></p><p>The Journal of Cell Biology 2008;182(2):301-313.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2483530.</p><p></p

Spatial Reorganization of the Endoplasmic Reticulum during Mitosis Relies on Mitotic Kinase Cyclin A in the Early <i>Drosophila</i> Embryo

<div><p>Mitotic cyclin-dependent kinase with their cyclin partners (cyclin:Cdks) are the master regulators of cell cycle progression responsible for regulating a host of activities during mitosis. Nuclear mitotic events, including chromosome condensation and segregation have been directly linked to Cdk activity. However, the regulation and timing of cytoplasmic mitotic events by cyclin:Cdks is poorly understood. In order to examine these mitotic cytoplasmic events, we looked at the dramatic changes in the endoplasmic reticulum (ER) during mitosis in the early <i>Drosophila</i> embryo. The dynamic changes of the ER can be arrested in an interphase state by inhibition of either DNA or protein synthesis. Here we show that this block can be alleviated by micro-injection of Cyclin A (CycA) in which defined mitotic ER clusters gathered at the spindle poles. Conversely, micro-injection of Cyclin B (CycB) did not affect spatial reorganization of the ER, suggesting CycA possesses the ability to initiate mitotic ER events in the cytoplasm. Additionally, RNAi-mediated simultaneous inhibition of all 3 mitotic cyclins (A, B and B3) blocked spatial reorganization of the ER. Our results suggest that mitotic ER reorganization events rely on CycA and that control and timing of nuclear and cytoplasmic events during mitosis may be defined by release of CycA from the nucleus as a consequence of breakdown of the nuclear envelope.</p></div

Arresting the <i>Drosophila</i> embryo in interphase maintains the ER in an interphase-like state.

<p>(<b>A</b>) Time-lapse confocal images of a Pdi-GFP (green) / H2-RFP (red) transgenic embryo injected at metaphase cycle 10 with the DNA replication inhibitor, aphidicolin (APH) and viewed during cycle 11. APH, arrests the embryo in S-phase of cycle 11. In the presence of APH, the ER displayed a loose uniform distribution around the nuclei denoting an interphase-like state. This interphase-like state of the ER persists for greater than 30 minutes without any changes to either localization or structure. This is quantified in the fluorescence intensity traces below (see yellow dotted-lines in merged images). H2-RFP signal inside the nucleus does not increase over this time period as well (arrowheads). (<b>B</b>) Time-lapse confocal images of a Pdi-GFP (green) / H2-RFP (red) embryo injected with the protein synthesis inhibitor cycloheximide (CHX) at metaphase of cycle 10 and viewed during interphase of the following cycle. Similar to APH, CHX induced arrest which maintained the ER in an interphase-like state. This is quantified below, as in A. (<b>C</b>) Similar background and approach as A and B. Embryos were injected with an APH+CHX cocktail. ER membrane maintained an interphase-like organization as seen in APH injections alone. Scale bar is 10 μm. Time is in min:sec.</p

3D reconstruction of ER structural changes display a clustering of extended cisternae at the spindle poles during metaphase.

<p>Embryos expressing Pdi-GFP (green) and H2-RFP (red) were fixed and imaged using confocal microscopy. (<b>A</b>) Upper panels (view 1) represent a top view of the nucleus and surrounding ER along the xy-plane and bottom panels (view 2) show the nucleus and ER ~45° -75° tilt along the in the y-plane. Embryos were imaged in the z-direction with a step size of 0.1 μm and subject to 3D reconstruction software. (<b>B</b>) At telophase of cycle 11, the ER is globular and spread along the reforming nuclear envelope and at the midbody (view 1, arrowhead). Exiting mitosis, at interphase, the ER is spread loosely through the cytoplasm outline the nuclear envelope. At prophase, the ER becomes defined and begins to cluster and propagate apically at the spindle poles. These clusters are not uniform in size and appear to be sheet-like structures (view 2, arrows). At metaphase, the clusters are found at the spindle poles and appear to be connected along the spindle area forming a sheath (view 1, arrow). In anaphase, ER cisternal clusters appear with the segregating chromosomes and at the midbody (view 2, arrow). Scale bar is 5 μm.</p

The ER displays dramatic structural and localization changes during mitosis in the early <i>Drosophila</i> embryo.

<p>(<b>A</b>) Mitotic ER dynamics were examined in cycle 11 transgenic <i>Drosophila</i> embryos expressing the ER marker Pdi-GFP and the DNA marker H2-RFP using time-lapse confocal microscopy. Phases of mitosis are listed at the top with relative time (min:sec) listed in the merge panels. During interphase, ER (green) was initially spread loosely around the nucleus. Upon entry into mitosis, ER accumulates around the nucleus and was rapidly converted to thick, perinuclear cisternae upon chromosome condensation (red) and prophase onset. in prometaphase, the ER membrane reorganizes with the developing mitotic spindle and begins to accumulate at the spindle poles. At metaphase and anaphase the ER is aligned with the mitotic spindle and displays movement towards the spindle poles (arrow). During late anaphase and telophase, the ER sees a rapid localization around the segregated, decondensing chromosomes and a localization at the central spindle / midbody (arrowhead). Scale bar is 10 μm. (<b>B</b>) High magnification of mitotic ER changes following a single nucleus used for quantification of ER movements shown in C (asterisk in A). Yellow line denotes fluorescence trace shown in C. Scale bar is 5 μm. (<b>C</b>) Fluorescence intensity trace of ER (green line) and chromosomes (red line) along 20 μm of the developing embryo. ER fluorescence is maximal just adjacent to the nuclear space, but is excluded from the nucleus. During interphase, the ER is evenly distributed throughout the cytoplasm. Intensity around the nucleus increases during mitosis and follows the extension of the spindle. Pdi-GFP signal intensity reached maximum during metaphase (arrows). Condensation and alignment of chromosomes at the metaphase plate are marked by the arrowhead. At telophase, two new nuclear envelopes are formed with a large peak at the remaining midbody.</p

Mitotic Cyc:CDK1 Activity is Necessary for Mitotic ER Dynamics

<p>(<b>A</b>) Cycle 11 transgenic embryo expressing Pdi-GFP / H2-RFP following simultaneous dsRNA-mediated knockdown of Cyclins A,B, and B3. When all three mitotic cyclins were knocked-down, there was a general arrest of the embryo prior to entry into mitosis and a block in ER spatial reorganization events. ER tubules persisted between adjacent nuclei. Chromosomes incompletely condensed (arrowhead) and the ER occasionally invaded the nuclear space (arrow). (<b>B</b>) Quantification of the induced arrest from injection of dsRNA directed at all three mitotic cylins shown in A. Intensities of Pdi-GFP and H2-RFP fluorescence are represented by green and red, respectively. (<b>C</b>) Arrest of ER membrane dynamics was further confirmed by examination of the ER shaping protein, Rtnl1. Injection of dsRNA directed at all three mitotic cyclins into a Rtnl1-GFP / H2-RFP embryo produced an arrest prior to mitotic entry, indicating that Rtnl1 is able to change localization independent of mitotic cyclin/CDK activity. (<b>D</b>) Quantification of arrest seen in C with Rtnl1-GFP in green and H2-RFP in red. Scale bars are 10 μm. Time is in min:sec.</p