Isolation and partial purification of the Saccharomyces cerevisiae cytokinetic apparatus - PubMed (original) (raw)

Isolation and partial purification of the Saccharomyces cerevisiae cytokinetic apparatus

Brian A Young et al. Cytoskeleton (Hoboken). 2010 Jan.

Abstract

Cytokinesis is the process by which a cell physically divides in two at the conclusion of a cell cycle. In animal and fungal cells, this process is mediated by a conserved set of proteins including actin, type II myosin, IQGAP proteins, F-BAR proteins, and the septins. To facilitate biochemical and ultrastructural analysis of cytokinesis, we have isolated and partially purified the Saccharomyces cerevisiae cytokinetic apparatus. The isolated apparatus contains all components of the actomyosin ring for which we tested-actin, myosin heavy and light chain, and IQGAP-as well as septins and the cytokinetic F-BAR protein, Hof1p. We also present evidence indicating that the actomyosin rings associated with isolated cytokinetic apparati may be contractile in vitro, and show preliminary electron microscopic imaging of the cytokinetic apparatus. This first successful isolation of the cytokinetic apparatus from a genetically tractable organism promises to make possible a deeper understanding of cytokinesis.

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Figures

Figure 1

Figure 1

Release of intact Myo1-GFP rings from S. cerevisiae cells. A & B) Isolated Myo1-GFP rings from S. cerevisiae. Rings were concentrated for visualization by 13,000xg pelleting (See Figure 2). A) Comparisions of long pass GFP (GFPLP) fluorescent images and bright field images of close-up views of rings. Bright field images show that rings are not associated with cells. B) whole camera field views of rings. Bars ∼ 1 μm (1032 nm).

Figure 2

Figure 2

Concentration and enrichment of the budding yeast cytokinetic apparatus. A & B) Differential subcellular fractionation of lysate (cleared of unlysed cells by 300_xg_ spin). In A) 10 μg of each fraction was assayed for Myo1-GFP by quantitative immunoblotting. In B) the number of intact Myo1-GFP rings pelleted in 13,000_xg_ and 100,000_xg_ fractions was measured by spinning 15 μg clarified lysate onto slides and counting rings using fluorescence microscopy. C & D) Discontinuous sucrose density gradient fractionation. In C) 25 μl of each fraction was assayed for Myo1p by quantitative immunoblotting. In D) fractions were assayed by pelleting Myo1-GFP rings onto slides and counting microscopically. In both B & D) only morphologically distinct rings (crisp fluorescent outline, closed figures where the outline does not cross itself) were counted. “Fields” refers to ocular fields using a 100× objective (Area= 38,013 μm2). Error bars represent standard error of the mean of three measurements of ring numbers.

Figure 3

Figure 3

Known components of the cytokinetic apparatus are associated with Myo1-GFP rings purified by sucrose gradient. Rings were purified from cells containing markers indicated to the left (yeast strain number is in parenthesis). A) Iqg1p association was tested by RFP fluorescence imaging. B) Actin association was tested by rhodamine-phalloidin staining. C) Mlc2p-myc association was tested by α-myc antibody staining. D) Hof1p-HA association was tested by α-HA antibody staining. In some cases Hof1p staining throughout rings was less uniform compared to other components. E) Cdc11p (septin) association was tested with affinity purified α-Cdc11p antibody staining. Percentage of rings where component in question was associated: Iqg1p 96% (24/25); Actin 92% (23/25); Mlc2p 100%(15/15); Hof1p 93% (14/15); Cdc11p 100% (15/15). Only morphologically distinct rings (crisp fluorescent outline, closed figures where the outline does not cross itself) were tested for association. Bars ∼1 μm (1032 nm).

Figure 4

Figure 4

Evidence for functionality of isolated cytokinetic apparatus. A) Number of Myo1-GFP rings was measured for isolated rings alone, rings incubated with ATP and cytokinetic extracts; and rings incubated with cytokinetic extract and a highly active ATPase, apyrase, for ATP depletion. Consistent with in vitro ring contraction, ring titers declined in an energy-dependent fashion. Incubation time was in excess of that required for full contraction of rings in vivo. Rings were titered by pelleting onto slides and counted by fluorescence microscopy. “Fields” here refers to ocular fields using a 100× objective. Only morphologically distinct rings were counted. B) Size distribution of isolated Myo1-GFP rings in the absence of activation by ATP and cytokinetic extract (i.e. 0 minutes contraction time) was measured for rings incubated with cytokinetic extract & apyrase. C) Size distribution of isolated rings after incubation with ATP and cytokinetic extract for approximately half the time necessary for ring contraction in vivo. In B & C) ring sizes were determined by photographing pelleted rings and measuring them using ImageJ software. “Fields” here refers to CCD camera fields using a 100× objective. In both B & C) 3 sets of 15 fields were measured. Error bars represent the standard error of the mean.

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