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First published online 28 April 2009
doi: 10.1242/jcs.044651

Journal of Cell Science 122, 1691-1699 (2009)
Published by The Company of Biologists 2009
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Ancient Leishmania coronin (CRN12) is involved in microtubule remodeling during cytokinesis

Amogh A. Sahasrabuddhe, Ramesh C. Nayak and Chhitar M. Gupta*

Division of Molecular and Structural Biology, Central Drug Research Institute, M.G. Marg, Lucknow 226001, India


Figure 1
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  		Fig. 1. Intracellular distribution of CRN12 and actin in resting and dividing Leishmania promastigotes. (A) Immunofluorescence images showing colocalization of CRN12 and actin at most of the places and a predominant localization of CRN12 in the flagellar pocket region in the resting cells. (B) Immunofluorescence images showing localization of both CRN12 and actin in the dividing cells. All these cells show a characteristic pattern of CRN12 accumulation at both the flagellar end and posterior pole. Arrowheads indicate posterior pole accumulation of CRN12 in the dividing cells. Ph, phase; Trans, transmission; Coro, CRN12; Act, actin. Scale bars: 5 µm.

 

Figure 2
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  		Fig. 2. Interactions of CRN12 with microtubules and kinesin K39. (A) Immunofluorescence images showing colocalization of CRN12 with positive ends of microtubules stained with YL1/2 antibodies. Unlike resting cells, the dividing cells show clear colocalization with microtubules (arrowheads) at their posterior poles. Scale bar: 5 µm. (B) Immunofluorescence images of resting and dividing cells labeled for CRN12 and kinesin K39. Arrows show colocalization of both the proteins in the flagellar pocket and posterior pole regions in the dividing cells. Notably, no such co-localization at the posterior pole in the resting cells is visible. Scale bars: 5 µm. (C) Western blot analysis showing presence of CRN12 in the microtubule cytoskeleton preparation. (a) Coomassie blue stained blot; (b) western blot of (a) probed with anti-CRN12 antibodies. Csk, detergent insoluble microtubule cytoskeleton; Sol, soluble cytosolic fraction; Mr, molecular weight markers. (D) Western blot analyses of tubulin and rK39 immunoprecipitates (IP) for the co-presence of CRN12. (a) Immunoprecipitate, obtained using anti-tubulin antibodies, probed with anti-CRN12 antibodies; (b) immunoprecipitate, obtained using anti-rK39 antibodies, probed with anti-CRN12 antibodies.

 

Figure 3
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  		Fig. 3. Localization of CRN12 in the basal body region marked by centrin in the NP-40-treated (0.5%, v/v) Coro+/+ cells. (A) Immunofluorescence images showing colocalization of CRN12 and centrin in the basal body region. Arrows indicate the colocalization. (B) Immunofluorescence image of a magnified cell cytoskeleton showing presence of CRN12 in the basal body region and its deviation from the flagellar fibrils stained with centrin antibodies. (C) Immunofluorescence image of the cytoskeleton stained for CRN12 and actin to show their different locations in the flagellar pocket region. Arrowheads indicate flagellar pocket regions of the promastigote cytoskeleton in the transmission images. Boxed regions are magnified further for more clarity. Arrows indicate the colocalization. Trans, transmission; Cen, centrin; Coro, CRN12. Scale bars: 5 µm.

 

Figure 4
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  		Fig. 4. (A) DNA constructs of Leishmania CRN12 gene deletion cassettes. Schematic representation of CRN12 locus in L. donovani before and after integration of Neo and/or Hyg cassettes, and incorporation of restriction sites BamHI (B), SalI (S), NotI (N) and HindIII (H). Sc indicates SacI sites in the genome adjacent to the CRN12 locus. Sites for primers P1-P4, used for the confirmation of Neo and Hyg cassette integration and amplification of CRN12 (arrowheads) by PCR, are also shown. (B) Western blot analysis of CRN12 mutants. Lanes 1-10, 10 Coro–/– clones showing varied levels of CRN12 expression. (C) Flow-cytometry analysis of Coro+/+, Coro+/– and Coro–/– cells showing similar DNA contents with 2.62%, 10.86% and 6.84% G2/M populations, respectively, as determined by ModFit program (version 3.0).

 

Figure 5
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  		Fig. 5. Effect of CRN12 depletion in Coro+/– Leishmania promastigotes on their morphology and growth. (A) Microscopic images of Coro+/+ and Coro+/– cells showing the presence of a significant number of bipolar cells in Coro+/– cell population. (a) Phase contrast; (b) DAPI-stained nuclei and kinetoplasts; (c) merge of `a' and `b'. Arrows indicate the bipolar cells; scale bars: 20 µm. (B) Growth curve of Coro+/+ (black triangles) and Coro+/– cells (white triangles) showing reduction of growth rate in the log phase and appearance of bipolar cells in heterozygous mutants (black circles, Coro+/+ cells; white circles, Coro+/– cells). Values shown are means of three independent experiments ± s.d.

 

Figure 6
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  		Fig. 6. Analyses of bipolar cell formation and cell growth after episomal complementation of CRN12 in Coro+/– cells. Black triangles and black circles indicate appearance of bipolar cells in p6.5MCS vector and p6.5MCS-CRN12 transfectants, respectively. Inset shows growth curve of p6.5MCS and p6.5MCS-CRN12 transfectants (white triangles and white circles, respectively), and western blot of both the transfectants showing increased CRN12 expression levels (2.16±0.26-fold, n=3) in p6.5MCS-CRN12 transfectants when compared with p6.5MCS transfectants. Densitometric values of CRN12 were normalized with actin (internal loading control).

 

Figure 7
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  		Fig. 7. Analysis of microtubule organization in the bipolar-cylindrical Coro+/– cells. (A) Labeling of microtubule positive ends by anti tyrosinated {alpha}-tubulin antibodies (rat monoclonal YL1/2). (a) Positive end labeling that specifically marks posterior pole of the resting and dividing Coro+/+ cells; (b) positive end clustering at different locations in Coro+/– cells with bipolar morphology. Scale bars: 5 µm. (B) Negatively stained transmission electron microscopic image of Triton X-100 treated Coro+/– cell with bipolar morphology, showing its microtubular organization. (a) Manually reconstituted electron micrograph of eight images taken at high magnification clearly showing continuity of corset-microtubules from one flagellar end to the other. (b,c) Negatively stained corset-microtubules showing their interlinking patterns in the control cell and bipolar cell, respectively. Arrows indicate sites of interlinking.

 

Figure 8
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  		Fig. 8. Localization of CRN12, tubulin, kinesin K39, centrin and actin in the Coro+/– cells. (A) Immunofluorescence images of a field showing different stages of the Coro+/– cells and distribution of CRN12 and positive ends of microtubules. Asterisks indicate bipolar cells, arrows indicate normally dividing cells and arrowheads indicate colocalizations of CRN12 and positive ends of microtubules. (B) Immunofluorescence images showing distribution of CRN12 and actin in bipolar cells. (C) Immunofluorescence images showing distribution of CRN12 and kinesin K39. These two proteins colocalize at various places but do not concentrate in the middle region of the bipolar cell. Arrowheads indicate colocalization. (D) Immunofluorescence images of bipolar cell cytoskeleton showing conserved locations of CRN12 in the basal body region marked by centrin. Arrowheads indicate colocalization. For clear presentation of CRN12 colocalization, images have been collected at slightly increased gain settings and are not quantitative.

 

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