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Organization of microtubule assemblies in Dictyostelium syncytia depends on the microtubule crosslinker, Ase1

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Abstract

It has long been known that the interphase microtubule (MT) array is a key cellular scaffold that provides structural support and directs organelle trafficking in eukaryotic cells. Although in animal cells, a combination of centrosome nucleating properties and polymer dynamics at the distal microtubule ends is generally sufficient to establish a radial, polar array of MTs, little is known about how effector proteins (motors and crosslinkers) are coordinated to produce the diversity of interphase MT array morphologies found in nature. This diversity is particularly important in multinucleated environments where multiple MT arrays must coexist and function. We initiate here a study to address the higher ordered coordination of multiple, independent MT arrays in a common cytoplasm. Deletion of a MT crosslinker of the MAP65/Ase1/PRC1 family disrupts the spatial integrity of multiple arrays in Dictyostelium discoideum, reducing the distance between centrosomes and increasing the intermingling of MTs with opposite polarity. This result, coupled with previous dynein disruptions suggest a robust mechanism by which interphase MT arrays can utilize motors and crosslinkers to sense their position and minimize overlap in a common cytoplasm.

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Acknowledgments

We appreciate the use of Wadsworth Center’s Molecular Genetics Core for DNA sequencing. We thank Dr. Wolfgang Nellen for kindly providing the GFP-CenH3 expression plasmid, Dr. Manfred Schliwa for the GFP-γ-tubulin construct, Valentin Magidson for general light microscopy assistance, and Dr. Vitali Sikirzhytski for help with statistical analysis. We are grateful to the efforts at http://dictybase.org to archive and annotate Dictyostelium sequence information. This work was supported in part by the NSF (MCB-1051612 to M.P.K, DBI-1062963 for REU support of K.I), and the NIH (GM59363 to A.K).

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Authors and Affiliations

  1. Division of Translational Medicine, NYS Department of Health, Wadsworth Center, Empire State Plaza, PO Box 509, Albany, NY, 12201-0509, USA

    Irina Tikhonenko, Karen Irizarry, Alexey Khodjakov & Michael P. Koonce

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  1. Irina Tikhonenko
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  2. Karen Irizarry
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Correspondence to Michael P. Koonce.

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18_2015_2026_MOESM1_ESM.tif

Supplemental Fig. 1. Targeted disruption of Ase1 isoforms by homologous recombination. The schematics in A diagram the strategy used to target insertion of a blasticidin S resistance cassette and disrupt gene transcription of Ase1A and Ase1B. Arrows show position of PCR primers used to screen clones for targeted inserts. Figure inserts show RT-PCR reactions in WT and KO strains. While ample message can be detected in WT cells, no gene products are seen in the Ase1A− or B− strains. Panel B shows growth curves of WT, Ase1A− and Ase1B− cells. (TIFF 2850 kb)

Supplementary material 2 (TIFF 2649 kb)

18_2015_2026_MOESM3_ESM.tif

Supplemental Fig. 2. Centromeres rapidly segregate during division in Ase1A− cells. Sequential frames (20 s interval) of a cell labeled with the centromere marker, GFP-DdCenH3 [51]. Centromere clusters separate here at a rate comparable to the fast centrosome movements quantified in Fig. 4. Bar = 5 μm. (TIFF 88 kb)

18_2015_2026_MOESM4_ESM.tif

Supplemental Fig. 3. Amplitude Spectrum of intercentrosome distance in WT vs. Ase1A− cells. Quantitation of movement between centrosome pairs, as presented in Fig. 6. Distance and frequency of 11 WT and 14 Ase1A− pairs were measured using a Fourier transform in a MATLAB environment. The trendlines are fitted to a cubic polynomial Eq. 1 px = 107 nm. (TIFF 2653 kb)

18_2015_2026_MOESM5_ESM.tif

Supplemental Fig. 4. Immunoblot of soluble Ase1A. Left panel shows Coomassie-stained PAGE lanes of supernatant and pellet following cell lysis and nuclear sedimentation. Middle and right panels show immunoblots from the same samples probed with an antibody against GFP. The left blot is from cells expressing the ~ 116 kDa GFP-Ase1A fusion; the right blot is from cells expressing ~ 95 kDa GFP-histone H3 like variant (Sup Fig. 2, [51]). The arrowhead points out a trace amount of Ase1A that is present in the supernatant, while the bulk of the polypeptide copellets with intact nuclei. The H3v1 samples are provided to control for potential nuclear breakage; no H3v1 fusion product is detected in the soluble fraction. (TIFF 10777 kb)

18_2015_2026_MOESM6_ESM.tif

Supplemental Fig. 5. MT disorganization in kinesin-4− cells. The left two panels show interphase binucleate cells where the Dd kinesin-4 family isoform (kif8) has been deleted [38]. Note a similar disorganization to the MT patterns as in Ase1A deletion. The two right panels show mitotic cells, demonstrating that a proper spindle forms in the absence of this kinesin. Bar = 10 μm (TIFF 495 kb)

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Tikhonenko, I., Irizarry, K., Khodjakov, A. et al. Organization of microtubule assemblies in Dictyostelium syncytia depends on the microtubule crosslinker, Ase1. Cell. Mol. Life Sci. 73, 859–868 (2016). https://doi.org/10.1007/s00018-015-2026-8

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