Like the editor said: "The textbook model, where the mitotic spindle is assumed to direct the position of the cleavage furrow, is not sufficient to explain furrow position during asymmetric cell division, at leas in the Drosophila neuroblasts. Instead, a novel pathway operates in which the Pins (Partner of Inscuteable) polarity complex polarizes furrow-forming proteins to the basal cortex where they induce contractile ring formation."
My friend told me this paper. And, it is quite interesting! Spinning disk on whole animal, very neat live imaging!
MARCM analysis
For generating Mira MARCM clones 40, we crossed the analysis line hsFLP70/hsFLP70; worGal4, UAS-Cherry:Jupiter, Sqh:GFP, tubGal80 FRT82B/TM6C, Sb (this work) to Mira FRT82B mirazz178 and heat-shocked the progeny 24–48 h after larval hatching for 1 h at 37 °C. For live imaging, mira mutant clones of third-instar larvae were used.
Pavarotti RNAi experiment
Pavarotti knockdown was achieved by crossing worGal4 (ref. 9) driver line to UAS-PavRNAi46137 (ref. 39). Loss of Pavarotti was confirmed using the anti-Pav antibody 16.
Colcemid experiments
For colcemid experiments, the following strains were used: +; worGal4, UAS-Cherry:Jupiter, Sqh:GFP (this work) or +; worGal4, UAS-Cherry:Jupiter, Sqh:GFP; rodH4.8 (this work).
Wild-type or rodH4.8 mutant neuroblasts were incubated with colcemid in live imaging medium 29 at a final concentration of 0.1 μm ml−1. Live imaging was started without delay. Mild spindle phenotypes became apparent immediately after colcemid exposure, whereas complete spindle depolymerization was seen approximately 30–60 min after colcemid addition.
Imaging, post-imaging procedures and measurements
Live imaging methods were previously described 29. Fixed preparations were imaged on a Leica SP2, and for Supplementary Fig. 1a on a Leica SP5, confocal microscope. Live samples were imaged on a McBain spinning disc confocal microscope equipped with a Hamamatsu EM-CCD (electron-multiplying charge-coupled device) camera, using a ×63 1.4 numerical aperture oil-immersion objective. Pixel intensity measurements (Fig. 1c, d) were performed using ImageJ. Only one-half of the neuroblasts’ cortex was measured starting at the apical cortex and ending at the basal cortex. Post-imaging processing and measurements were performed in ImageJ or Imaris 6.2–7.0 (Bitplane).
Larval staging
For all experiments, late second- or third-instar larvae were used for analysis.
Nature. 2010 Sep 2;467(7311):91-4.
A spindle-independent cleavage furrow positioning pathway.
Cabernard C, Prehoda KE, Doe CQ.[1] Howard Hughes Medical Institute, University of Oregon, Eugene, Oregon 97403, USA [2] Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA [3] Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403, USA.
The mitotic spindle determines the cleavage furrow site during metazoan cell division, but whether other mechanisms exist remains unknown. Here we identify a spindle-independent mechanism for cleavage furrow positioning in Drosophila neuroblasts. We show that early and late furrow proteins (Pavarotti, Anillin, and Myosin) are localized to the neuroblast basal cortex at anaphase onset by a Pins cortical polarity pathway, and can induce a basally displaced furrow even in the complete absence of a mitotic spindle. Rotation or displacement of the spindle results in two furrows: an early polarity-induced basal furrow and a later spindle-induced furrow. This spindle-independent cleavage furrow mechanism may be relevant to other highly polarized mitotic cells, such as mammalian neural progenitors.
A very interesting point
* More informative were the approximately 11% of pins mutant neuroblasts that had a symmetric spindle and divided symmetrically; all of these neuroblasts lacked Pav/Myosin basal cortical enrichment, lacked basal furrows and never formed 'polar lobes'
* To increase the percentage of symmetrically dividing pins mutant neuroblasts, we combined pins with a mutation in dlg, which is required for normal spindle asymmetry 9. We found that 100% of the dlg;;pins double mutant neuroblasts showed symmetric spindles, and they all lacked Myosin basal cortical enrichment and basally displaced furrows
* To test if Dlg has a role in the polarity induced furrow pathway, we examined dlg single mutants. Only a small fraction had a symmetric spindle (6%, n = 65); of these neuroblasts, two exhibited basally enriched Myosin (data not show) and two showed symmetric cortical Myosin
In neuroblasts these pathways appear to work partly redundantly.
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