Sonja Stenmark

Deciphering the rules governing assembly order of mammalian septin complexes

Vertebrates express 9–17 septin family members known to oligomerize into diverse structures, but their native assembly states have remained elusive. The results presented suggest a generic model for how the temporal order of septin assembly directs the subunit arrangement within distinct pools of six- to eight-subunit core heteromers.

Differential functional interplay of TOGp/XMAP215 and the KinI kinesin MCAK during interphase and mitosis.

XMAP215/TOGp family members and KinI kinesins are conserved microtubule (MT)‐regulatory proteins, and have been viewed as possessing prominent antagonistic stabilizing/destabilizing activities that must be balanced. Here, interdependencies between TOGp and the KinI kinesin MCAK were analyzed in human leukemia cells. A system was established that permits inducible overexpression in homogeneous cell populations that simultaneously synthesize interfering short hairpin RNAs. We present evidence that the functional interplay of TOGp and MCAK proteins is manifested as three distinct phenotypes during the cell cycle. The first involves a role for TOGp in protecting spindle MTs from MCAK activity at the centrosome, which appears essential to prevent the formation of disorganized multipolar spindles. The second phenotype involves TOGp‐dependent counteraction of excessive MCAK activity during mitosis, which recapitulates the previously established plus‐end specific counteractive activities in vitro. The third involves an unexpected destabilization of the interphase MTs by overexpressed TOGp, a phenotype that requires endogenous MCAK. We hypothesize that TOGp‐dependent prevention of MCAK‐mediated spindle disorganization, as evidenced by depletion experiments, reflects a primary physiological role for TOGp in human somatic cells.

CaMKIIγ‐mediated inactivation of the Kin I kinesin MCAK is essential for bipolar spindle formation

MCAK, a member of the kinesin‐13 family, is a microtubule (MT) depolymerase that is necessary to ensure proper kinetochore MT attachment during spindle formation. Regulation of MCAK activity and localization is controlled in part by Aurora B kinase at the centromere. Here we analyzed human cells depleted of the ubiquitous Ca2+/calmodulin‐dependent protein kinase IIγ isoform (CaMKIIγ) by RNA interference and found that CaMKIIγ was necessary to suppress MCAK depolymerase activity in vivo. A functional overlap with TOGp, a MT regulator known to counteract MCAK, was suggested by similar CaMKIIγ‐ and TOGp‐depletion phenotypes, namely disorganized multipolar spindles. A replicating vector system, which permits inducible overexpression in cells that simultaneously synthesize interfering short hairpin RNAs, was used to dissect the functional interplay between CaMKIIγ, TOGp, and MCAK. Our results revealed two distinct but functionally overlapping mechanisms for negative regulation of the cytosolic/centrosomal pool of MCAK. These two mechanisms, involving CaMKIIγ and TOGp, respectively, are both essential for spindle bipolarity in a normal physiological context, but not in MCAK‐depleted cells.

Microtubules support a disk-like septin arrangement at the plasma membrane of mammalian cells

Septin assemblies during the interphase of animal cells remain poorly defined and are the topic of this report. The data point to a general model for assembly of higher-order septin arrangements at locations providing the greatest opportunity for binding cooperativity, which depends on both the cell type and external cues.

Global regulation of the interphase microtubule system by abundantly expressed Op18/stathmin.

Op18/stathmin (Op18), a conserved microtubule-depolymerizing and tubulin heterodimer-binding protein, is a major interphase regulator of tubulin monomer-polymer partitioning in diverse cell types in which Op18 is abundant. Here, we addressed the question of whether the microtubule regulatory function of Op18 includes regulation of tubulin heterodimer synthesis. We used two human cell model systems, K562 and Jurkat, combined with strategies for regulatable overexpression or depletion of Op18. Although Op18 depletion caused extensive overpolymerization and increased microtubule content in both cell types, we did not detect any alteration in polymer stability. Interestingly, however, we found that Op18 mediates positive regulation of tubulin heterodimer content in Jurkat cells, which was not observed in K562 cells. By analysis of cells treated with microtubule-poisoning drugs, we found that Jurkat cells regulate tubulin mRNA levels by a posttranscriptional mechanism similarly to normal primary cells, whereas this mechanism is nonfunctional in K562 cells. We present evidence that Op18 mediates posttranscriptional regulation of tubulin mRNA in Jurkat cells through the same basic autoregulatory mechanism as microtubule-poisoning drugs. This, combined with potent regulation of tubulin monomer-polymer partitioning, enables Op18 to exert global regulation of the microtubule system.

Mammalian SEPT9 isoforms direct microtubule-dependent arrangements of septin core heteromers

Cell type–specific alternative splicing results in six confirmed mammalian SEPT9 isoforms. SEPT9 expression levels dictate the hexamer-to-octamer ratio of septin core heteromers, and isoform compositions and expression levels together determine higher-order arrangements of septin filaments.

Cell type–specific expression of SEPT3-homology subgroup members controls the subunit number of heteromeric septin complexes

Heteromeric septin complexes serve as building blocks of filaments that organize the cortex of fungal and animal cells. This report addresses determinants that direct hetero-oligomerization of the 13 septin paralogues of mammals. It shows that three distinct septins direct assembly of tissue-specific heterotetramers.

Op18/Stathmin counteracts the activity of overexpressed tubulin-disrupting proteins in a human leukemia cell line

Op18/stathmin (Op18) is a phosphorylation-regulated and differentially expressed microtubule-destabilizing protein in animal cells. Op18 regulates tubulin monomer-polymer partitioning of the interphase microtubule system and forms complexes with tubulin heterodimers. Recent reports have shown that specific tubulin-folding cofactors and related proteins may disrupt tubulin heterodimers. We therefore investigated whether Op18 protects unpolymerized tubulin from such disruptive activities. Our approach was based on inducible overexpression of two tubulin-disrupting proteins, namely TBCE, which is required for tubulin biogenesis, and E-like, which has been proposed to regulate tubulin turnover and microtubule stability. Expression of either of these proteins was found to cause a rapid degradation of both alpha-tubulin and beta-tubulin subunits of unpolymerized, but not polymeric, tubulin heterodimers. We found that depletion of Op18 by means of RNA interference increased the susceptibility of tubulin to TBCE or E-like mediated disruption, while overexpressed Op18 exerted a tubulin-protective effect. Tubulin protection was shown to depend on Op18 levels, binding affinity, and the partitioning between tubulin monomers and polymers. Hence, the present study reveals that Op18 at physiologically relevant levels functions to preserve the integrity of tubulin heterodimers, which may serve to regulate tubulin turnover rates.