[Show abstract][Hide abstract] ABSTRACT: The role of centrosomes and centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays--termed an amphiaster ("a star on both sides")--that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB). Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC reassembled, and, prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ~35% of aMTOCs failed to split and separate before NEB, and these entered mitosis with persistent monastral spindles. Chromatin-associated RAN-GTP--the small GTPase Ran in its GTP bound state--could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but, in its absence, the fidelity of bipolar spindle assembly is highly compromised.
Current biology: CB 03/2011; 21(7):598-605. DOI:10.1016/j.cub.2011.02.049 · 9.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Sac1 PIP phosphatase is an enigmatic enzyme in that it occupies an intracellular location (ER) that is not normally associated with PIP signaling. Yet, genetic experiments in mice and silencing experiments in cultured cells report an essential housekeeping function for this protein. Detailed cellular analyses report maintenance of proper organization of the Golgi system, and of the mitotic spindle apparatus, are compromised when Sac1 functional thresholds are breached. While the Golgi derangements do not obviously affect protein transport through the organelle, the mitotic defects result in defects in progression through the G2/M stage of the cell cycle. Finally, both the catalytic PIP phosphatase activity, and its ability to be recycled back to the ER, represent essential functional features of the Sac1 enzyme. We expect that current insights for Sac1 will set the blueprint for future analyses of its functions. Many questions remain to be answered in this field: does Sac1 have important roles in ER and plasma membranes connections since ySac1 only degrades the plasma membrane-localized PtdIns 4-OH kinase Stt4 generated PtdIns-4-P? Does the ER-localized Sac1 play critical roles in regulating nuclear PIP signaling since ER is continuous with the nuclear envelope, and does it specifically happen in a certain stage during cell cycle progression? Does anchoring the Sac1-catalytic domain to other cellular membranes affect its function and what effects will be generated by mislocalizing the Sac1-catalytic domain to exotic membrane locations? Does the Golgi dispersion phenotype herald a crosstalk of the organelle with the mitotic apparatus (i.e. does Golgi disorganization provide ectopic nucleation sites for the γ-tubulin ring complex)? Clearly, there is much to be learned regarding the biological functions of Sac1-like lipid phosphatases, and we anticipate the discoveries yet to come will rival those derived from studies of the kinases - both in impact and in scope.
Advances in enzyme regulation 02/2009; 49(1). DOI:10.1016/j.advenzreg.2009.01.006
[Show abstract][Hide abstract] ABSTRACT: Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-alpha tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible-but before NEB is complete-results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation.
Cell Motility and the Cytoskeleton 08/2008; 65(8):595-613. DOI:10.1002/cm.20283 · 4.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phosphoinositides (PIPs) are ubiquitous regulators of signal transduction events in eukaryotic cells. PIPs are degraded by various enzymes, including PIP phosphatases. The integral membrane Sac1 phosphatases represent a major class of such enzymes. The central role of lipid phosphatases in regulating PIP homeostasis notwithstanding, the biological functions of Sac1-phosphatases remain poorly characterized. Herein, we demonstrate that functional ablation of the single murine Sac1 results in preimplantation lethality in the mouse and that Sac1 insufficiencies result in disorganization of mammalian Golgi membranes and mitotic defects characterized by multiple mechanically active spindles. Complementation experiments demonstrate mutant mammalian Sac1 proteins individually defective in either phosphoinositide phosphatase activity, or in recycling of the enzyme from the Golgi system back to the endoplasmic reticulum, are nonfunctional proteins in vivo. The data indicate Sac1 executes an essential household function in mammals that involves organization of both Golgi membranes and mitotic spindles and that both enzymatic activity and endoplasmic reticulum localization are important Sac1 functional properties.
Molecular biology of the cell 08/2008; 19(7):3080-96. DOI:10.1091/mbc.E07-12-1290 · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin-independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.
The Journal of Cell Biology 06/2008; 181(4):595-603. DOI:10.1083/jcb.200711160 · 9.69 Impact Factor