Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome
Karyn M. Austin, Mohan L. Gupta Jr., Scott A. Coats, Asmin Tulpule, Gustavo Mostoslavsky, Alejandro B. Balazs, Richard C. Mulligan, George Daley, David Pellman, Akiko Shimamura
Karyn M. Austin, Mohan L. Gupta Jr., Scott A. Coats, Asmin Tulpule, Gustavo Mostoslavsky, Alejandro B. Balazs, Richard C. Mulligan, George Daley, David Pellman, Akiko Shimamura
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Research Article Hematology

Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome

Abstract

Deficiencies in the SBDS gene result in Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome associated with leukemia predisposition. SBDS encodes a highly conserved protein previously implicated in ribosome biogenesis. Using human primary bone marrow stromal cells (BMSCs), lymphoblasts, and skin fibroblasts, we show that SBDS stabilized the mitotic spindle to prevent genomic instability. SBDS colocalized with the mitotic spindle in control primary BMSCs, lymphoblasts, and skin fibroblasts and bound to purified microtubules. Recombinant SBDS protein stabilized microtubules in vitro. We observed that primary BMSCs and lymphoblasts from SDS patients exhibited an increased incidence of abnormal mitoses. Similarly, depletion of SBDS by siRNA in human skin fibroblasts resulted in increased mitotic abnormalities and aneuploidy that accumulated over time. Treatment of primary BMSCs and lymphoblasts from SDS patients with nocodazole, a microtubule destabilizing agent, led to increased mitotic arrest and apoptosis, consistent with spindle destabilization. Conversely, SDS patient cells were resistant to taxol, a microtubule stabilizing agent. These findings suggest that spindle instability in SDS contributes to bone marrow failure and leukemogenesis.

Authors

Karyn M. Austin, Mohan L. Gupta Jr., Scott A. Coats, Asmin Tulpule, Gustavo Mostoslavsky, Alejandro B. Balazs, Richard C. Mulligan, George Daley, David Pellman, Akiko Shimamura

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Figure 3

SBDS binds to and stabilizes microtubules in vitro.

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SBDS binds to and stabilizes microtubules in vitro. (A) Endogenous SBDS ...
(A) Endogenous SBDS co-pellets with microtubules from cell lysates. Increasing concentrations of purified, taxol-stabilized bovine microtubules were incubated with HeLa cell extract, layered over a glycerol cushion, and centrifuged to pellet the microtubules and associated proteins. The pellets were analyzed by western blot for tubulin and SBDS. (B) Recombinant SBDS co-sediments with microtubules. Left: Coomassie-stained gel loaded with standard molecular markers (lane 1) or 3 μg purified recombinant SBDS protein (lane 2). Upper right: The fraction of 500 nM SBDS remaining in the supernatant (s) or co-pelleting (p) with increasing amounts of taxol-stabilized microtubules after centrifugation through a glycerol cushion was monitored by SBDS immunoblotting. Lower right: The average percentage bound from 3 experiments. The 1-site binding isotherm and Kd = 7.6 ± 3.0 μM were obtained by best-fit nonlinear regression using Graphpad Prism software. Approximately 14% of purified SBDS was co-pelleted with microtubules. Error bars represent SEM. (C) SBDS stabilizes microtubules in vitro. Preformed fluorescence-associated microtubules were diluted into PBS buffer alone or into buffer containing either taxol or increasing concentrations of purified, bacterially expressed recombinant SBDS protein. SBDS concentrations of 1.74 μM, 3.5 μM, and 7 μM were assayed. Dilution-induced depolymerization was monitored by the loss of microtubule-stimulated fluorescence signal. Three independent experiments yielded similar results. A representative experiment is shown. As a negative control, purified bovine serum albumin protein had no effect on microtubule stability (Supplemental Figure 4).