Elevated levels of VCA0117 (VasH) in response to external signals activate the type VI secretion system of Vibrio cholerae O1 El Tor A1552.

The type VI nanomachine is critical for Vibrio cholerae to establish infections and to thrive in niches co-occupied by competing bacteria. The genes for the type VI structural proteins are encoded in one large and two small auxiliary gene clusters. VCA0117 (VasH) - a σ54 -transcriptional activator - is strictly required for functionality of the type VI secretion system since it controls production of the structural protein Hcp. While some strains constitutively produce a functional system, others do not and require specific growth conditions of low temperature and high osmolarity for expression of the type VI machinery. Here, we trace integration of these regulatory signals to the promoter activity of the large gene cluster in which many components of the machinery and VCA0117 itself are encoded. Using in vivo and in vitro assays and variants of VCA0117, we show that activation of the σ54 -promoters of the auxiliary gene clusters by elevated VCA0117 levels are all that is required to overcome the need for specialized growth conditions. We propose a model in which signal integration via the large operon promoter directs otherwise restrictive levels of VCA0117 that ultimately dictates a sufficient supply of Hcp for completion of a functional type VI secretion system.

The Swedish COVID-19 approach: a scientific dialogue on mitigation policies.

During the COVID-19 pandemic, Sweden was among the few countries that did not enforce strict lockdown measures but instead relied more on voluntary and sustainable mitigation recommendations. While supported by the majority of Swedes, this approach faced rapid and continuous criticism. Unfortunately, the respectful debate centered around scientific evidence often gave way to mudslinging. However, the available data on excess all-cause mortality rates indicate that Sweden experienced fewer deaths per population unit during the pandemic (2020-2022) than most high-income countries and was comparable to neighboring Nordic countries through the pandemic. An open, objective scientific dialogue is essential for learning and preparing for future outbreaks.

Upregulated Op18/stathmin activity causes chromosomal instability through a mechanism that evades the spindle assembly checkpoint.

Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18-->E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis, conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability.

Predominant regulators of tubulin monomer-polymer partitioning and their implication for cell polarization.

The microtubule-system organizes the cytoplasm during interphase and segregates condensed chromosomes during mitosis. Four unrelated conserved proteins, XMAP215/Dis1/TOGp, MCAK, MAP4 and Op18/stathmin, have all been implicated as predominant regulators of tubulin monomer-polymer partitioning in animal cells. However, while studies employing the Xenopus egg extract model system indicate that the partitioning is largely governed by the counteractive activities of XMAP215 and MCAK, studies of human cell lines indicate that MAP4 and Op18 are the predominant regulators of the interphase microtubule-array. Here, we review functional interplay of these proteins during interphase and mitosis in various cell model systems. We also review the evidence that MAP4 and Op18 have interphase-specific, counteractive and phosphorylation-inactivated activities that govern tubulin subunit partitioning in many mammalian cell types. Finally, we discuss evidence indicating that partitioning regulation by MAP4 and Op18 may be of significance to establish cell polarity.

Interphase-specific phosphorylation-mediated regulation of tubulin dimer partitioning in human cells.

The microtubule cytoskeleton is differentially regulated by a diverse array of proteins during interphase and mitosis. Op18/stathmin (Op18) and microtubule-associated protein (MAP)4 have been ascribed opposite general microtubule-directed activities, namely, microtubule destabilization and stabilization, respectively, both of which can be inhibited by phosphorylation. Here, using three human cell models, we depleted cells of Op18 and/or MAP4 by expression of interfering hairpin RNAs and we analyzed the resulting phenotypes. We found that the endogenous levels of Op18 and MAP4 have opposite and counteractive activities that largely govern the partitioning of tubulin dimers in the microtubule array at interphase. Op18 and MAP4 were also found to be the downstream targets of Ca(2+)- and calmodulin-dependent protein kinase IV and PAR-1/MARK2 kinase, respectively, that control the demonstrated counteractive phosphorylation-mediated regulation of tubulin dimer partitioning. Furthermore, to address mechanisms regulating microtubule polymerization in response to cell signals, we developed a system for inducible gene product replacement. This approach revealed that site-specific phosphorylation of Op18 is both necessary and sufficient for polymerization of microtubules in response to the multifaceted signaling event of stimulation of the T cell antigen receptor complex, which activates several signal transduction pathways.

The Schistosoma mansoni protein Sm16/SmSLP/SmSPO-1 assembles into a nine-subunit oligomer with potential To inhibit Toll-like receptor signaling.

The Sm16/SmSLP/SmSPO-1 (Sm16) protein is secreted by the parasite Schistosoma mansoni during skin penetration and has been ascribed immunosuppressive activities. Here we describe the strategy behind the design of a modified Sm16 protein with a decreased aggregation propensity, thus facilitating the expression and purification of an Sm16 protein that is soluble in physiological buffers. The Stokes radii and sedimentation coefficients of recombinant and native proteins indicate that Sm16 is an approximately nine-subunit oligomer. Analysis of truncated Sm16 derivatives showed that both oligomerization and binding to the plasma membrane of human cells depend on multiple C-terminal regions. For analysis of immunomodulatory activities, Sm16 was expressed in Pichia pastoris to facilitate the preparation of a pyrogen/endotoxin-free purified protein. Recombinant Sm16 was found to have no effect on T-lymphocyte activation, cell proliferation, or the basal level of cytokine production by whole human blood or monocytic cells. However, Sm16 exerts potent inhibition of the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and poly(I:C) while being less efficient at inhibiting the response to the TLR ligand peptidoglycan or a synthetic lipopeptide. Since Sm16 specifically inhibits the degradation of the IRAK1 signaling protein in LPS-stimulated monocytes, our findings indicate that inhibition is exerted proximal to the TLR complex.

Aneugenic activity of Op18/stathmin is potentiated by the somatic Q18-->e mutation in leukemic cells.

Op18/stathmin (Op18) is a phosphorylation-regulated microtubule destabilizer that is frequently overexpressed in tumors. The importance of Op18 in malignancy was recently suggested by identification of a somatic Q18-->E mutation of Op18 in an adenocarcinoma. We addressed the functional consequences of aberrant Op18 expression in leukemias by analyzing the cell cycle of K562 cells either depleted of Op18 by expression of interfering hairpin RNA or induced to express wild-type or Q18E substituted Op18. We show here that although Op18 depletion increases microtubule density during interphase, the density of mitotic spindles is essentially unaltered and cells divide normally. This is consistent with phosphorylation-inactivation of Op18 during mitosis. Overexpression of wild-type Op18 results in aneugenic activities, manifest as aberrant mitosis, polyploidization, and chromosome loss. One particularly significant finding was that the aneugenic activity of Op18 was dramatically increased by the Q18-->E mutation. The hyperactivity of mutant Op18 is apparent in its unphosphorylated state, and this mutation also suppresses phosphorylation-inactivation of the microtubule-destabilizing activity of Op18 without any apparent effect on its phosphorylation status. Thus, although Op18 is dispensable for mitosis, the hyperactive Q18-->E mutant, or overexpressed wild-type Op18, exerts aneugenic effects that are likely to contribute to chromosomal instability in tumors.

MAP4 counteracts microtubule catastrophe promotion but not tubulin-sequestering activity in intact cells.

Microtubules are polar polymers that continually switch between phases of elongation and shortening, a property referred to as dynamic instability. The ubiquitous microtubule associated protein 4 (MAP4) shows rescue-promoting activity during in vitro assembly of microtubules (i.e., promotes transitions from shortening to elongation), but its regulatory role in intact cells is poorly defined. Here, we demonstrate that ectopic MAP4 promotes outgrowth of extended MTs during beta1-integrin-induced cell spreading. An inducible cotransfection protocol was employed to further analyze the regulatory role of MAP4 in human leukemia cells with microtubules partially destabilized by either ectopic tubulin-sequestering proteins or proteins that promote catastrophes (i.e., transitions from elongation to shortening). Coexpression of proteins that sequester free tubulin heterodimers with different efficiencies was found to abolish microtubule stabilization by MAP4. In contrast, however, the microtubule-stabilizing activity of MAP4 was found to suppress the activities of two distinct and specific catastrophe promoters, namely, XKCM1 and a nonsequestering truncation derivative of Op18/stathmin. These observations reveal specificity in the microtubule-stabilizing activity of MAP4 that differentiates between two mechanistically distinct types of MT destabilization.

The Schistosoma mansoni protein Sm16/SmSLP/SmSPO-1 is a membrane-binding protein that lacks the proposed microtubule-regulatory activity.

Sm16/SmSLP/SPO-1 (Sm16) has been identified as a developmentally regulated protein that is released from specific glands of the Schistosoma mansoni parasite during skin penetration. Sm16 has been ascribed both anti-inflammatory activities and a functional similarity with the conserved cytosolic tubulin-binding protein stathmin/Op18. Here we used a cell line to confirm signal peptide-dependent secretion and to define the secreted form of Sm16 for production in E. coli. We present evidence from both in vitro experiments and studies on transfected human cells that refute any functional similarity with stathmin/Op18. Instead of an Op18-like activity, we found that targeting of Sm16 to the cytosol of human cells, which was achieved by ectopic expression of Sm16 lacking the signal peptide, results in a caspase-dependent apoptotic response. Interestingly, by analysis of recombinant preparations we found that the secreted form of Sm16 is a lipid bilayer-binding protein that efficiently binds to the surface of diverse cell types by a polyanion-independent mechanism, which results in uptake by endocytosis. While the significance of the pro-apoptotic activity exerted by cytosolic Sm16 remains unclear, the present findings on cell-surface-binding properties of Sm16 seems likely to be of functional relevance during skin penetration of the parasite.

Deciphering the cellular functions of the Op18/Stathmin family of microtubule-regulators by plasma membrane-targeted localization.

The Op18/stathmin family of microtubule regulators includes the ubiquitous cytosolic Op18/stathmin (Op18) and the neuronal, primarily Golgi-associated proteins SCG10 and RB3, which all form ternary complexes with two head-to-tail-aligned tubulin heterodimers. To understand the physiological significance of previously observed differences in ternary complex stability, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. Herein, we show that, in contrast to constitutively active CD2-Op18-tetraA, both the CD2-SCG10 and CD2-RB3 chimeras sequestered tubulin at the plasma membrane, which results in >35% reduction of cytosolic tubulin heterodimer levels and consequent delayed formation of mitotic spindles. However, all three CD2 chimeras, including the tubulin sequestration-incompetent CD2-Op18-tetraA, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during interphase, but not during mitosis, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules. However, the differences in tubulin binding observed in cells also indicate conceptual differences between the functions of low-abundance neural family members, which will accumulate tubulin at specific cellular compartments, and the abundant cytosolic Op18 protein, which will not.

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

Septins are filament-forming proteins important for organizing the cortex of animal and fungal cells. In mammals, 13 septin paralogues were recently shown to assemble into core heterohexamer and heterooctamer complexes, which serve as building blocks for apolar filamentous structures that differ among cell types. To determine how tissue-specific septin paralogue expression may shape core heteromer repertoires and thereby modulate properties of septin filaments, we devised protocols to analyze native septin heteromers with distinct numbers of subunits. Our evidence based on genetically manipulated human cells supports and extends recent concepts of homology subgroup-restricted assembly into distinct categories of apolar heterohexamers and heterooctamers. We also identify a category of tetramers that have a subunit composition equivalent to an octameric building block. These atypical tetramers are prevalent in lymphocytes and neural tissues, in which octamers are abundant but hexamers are rare. Our results can be explained by tissue-specific expression of SEPT3 subgroup members: SEPT3, SEPT9, and SEPT12. These serve as cognate subunits in either heterooctamers or atypical tetramers but exhibit different preferences in various tissues. The identified tissue-specific repertoires of septin heteromers provide insights into how higher-order septin structures with differential properties and stabilities may form in diverse animal cell types.

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

Septin-family proteins assemble into rod-shaped heteromeric complexes that form higher-order arrangements at the cell cortex, where they serve apparently conserved functions as diffusion barriers and molecular scaffolds. There are 13 confirmed septin paralogues in mammals, which may be ubiquitous or tissue specific. Septin hetero-oligomerization appears homology subgroup directed, which in turn determines the subunit arrangement of six- to eight-subunit core heteromers. Here we address functional properties of human SEPT9, which, due to variable mRNA splicing, exists as multiple isoforms that differ between tissues. Myeloid K562 cells express three SEPT9 isoforms, all of which have an equal propensity to hetero-oligomerize with SEPT7-containing hexamers to generate octameric heteromers. However, due to limiting amounts of SEPT9, K562 cells contain both hexameric and octameric heteromers. To generate cell lines with controllable hexamer-to-octamer ratios and that express single SEPT9 isoforms, we developed a gene product replacement strategy. By this means we identified SEPT9 isoform-specific properties that either facilitate septin heteromer polymerization along microtubules or modulate the size range of submembranous septin disks-a prevalent septin structure in nonadhered cells. Our findings show that the SEPT9 expression level directs the hexamer-to-octamer ratio, and that the isoform composition and expression level together determine higher-order arrangements of septins.

Deciphering the rules governing assembly order of mammalian septin complexes.

Septins are conserved GTP-binding proteins that assemble into lateral diffusion barriers and molecular scaffolds. Vertebrate genomes contain 9-17 septin genes that encode both ubiquitous and tissue-specific septins. Expressed septins may assemble in various combinations through both heterotypic and homotypic G-domain interactions. However, little is known regarding assembly states of mammalian septins and mechanisms directing ordered assembly of individual septins into heteromeric units, which is the focus of this study. Our analysis of the septin system in cells lacking or overexpressing selected septins reveals interdependencies coinciding with previously described homology subgroups. Hydrodynamic and single-particle data show that individual septins exist solely in the context of stable six- to eight-subunit core heteromers, all of which contain SEPT2 and SEPT6 subgroup members and SEPT7, while heteromers comprising more than six subunits also contain SEPT9. The combined data suggest a generic model for how the temporal order of septin assembly is homology subgroup-directed, which in turn determines the subunit arrangement of native heteromers. Because mammalian cells normally express multiple members and/or isoforms of some septin subgroups, our data also suggest that only a minor fraction of native heteromers are arranged as perfect palindromes.

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

Septin family proteins oligomerize through guanosine 5'-triphosphate-binding domains into core heteromers, which in turn polymerize at the cleavage furrow of dividing fungal and animal cells. Septin assemblies during the interphase of animal cells remain poorly defined and are the topic of this report. In this study, we developed protocols for visualization of authentic higher-order assemblies using tagged septins to effectively replace the endogenous gene product within septin core heteromers in human cells. Our analysis revealed that septins assemble into microtubule-supported, disk-like structures at the plasma membrane. In the absence of cell substrate adhesion, this is the predominant higher-order arrangement in interphase cells and each of the seven to eight septin family members expressed by the two analyzed cell types appears equally represented. However, studies of myeloid and lymphoid cell model systems revealed cell type-specific alterations of higher-order septin arrangements in response to substrate adhesion. Live-cell observations suggested that all higher-order septin assemblies are mutually exclusive with plasma membrane regions undergoing remodeling. The combined data point to a mechanism by which densely arranged cortical microtubules, which are typical for nonadhered spherical cells, support plasma membrane-bound, disk-like septin assemblies.

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.

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.

CaMKIIgamma-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 Ca(2+)/calmodulin-dependent protein kinase IIgamma isoform (CaMKIIgamma) by RNA interference and found that CaMKIIgamma 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 CaMKIIgamma- 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 CaMKIIgamma, 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 CaMKIIgamma and TOGp, respectively, are both essential for spindle bipolarity in a normal physiological context, but not in MCAK-depleted cells.

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.

Interphase and monoastral-mitotic phenotypes of overexpressed MAP4 are modulated by free tubulin concentrations.

The microtubule-associated protein 4 (MAP4) has recently been shown to counteract destabilization of interphase microtubules caused by catastrophe promotion but not by tubulin sequestering. To address how MAP4 discriminates between destabilization of microtubules by these two mechanisms, we have evaluated the combined phenotypes of MAP4 coexpressed with Op18/stathmin family member derivatives with either catastrophe-promoting or sequestering activities. This approach relies on the finding that overexpression of MAP4 alone stabilizes microtubules during all phases of the cell cycle in human leukemia cells, and causes a potent mitotic block and a dramatic, previously unobserved, phenotype characterized by large monoastral spindles. Coexpression of either catastrophe-promoting or tubulin-sequestration-specific Op18 derivatives was found to modulate the activity of ectopic MAP4 during mitosis, but with differential functional outcome. Interestingly, the tubulin-sequestering derivative suppressed the monoastral mitotic phenotype of MAP4 (i.e. coexpression facilitated the formation of functional spindles). To evaluate whether this phenotypic suppression could be explained by tubulin-sequestration-dependent modulation of MAP4 activity, a plasma-membrane-targeted, tubulin-sequestering chimera was constructed to decrease the cytosolic free tubulin concentration substantially. This chimera likewise suppressed the monoastral phenotype caused by overexpression of MAP4, suggesting a direct downregulation of MAP4 activity by reduced free tubulin concentrations.

Molecular dissection of GTP exchange and hydrolysis within the ternary complex of tubulin heterodimers and Op18/stathmin family members.

The ubiquitous Op18 and the neural RB3 and SCG10 proteins are members of the oncoprotein18/stathmin family of microtubule regulators. These proteins bind two tubulin heterodimers via two imperfect helical repeats to form a complex of heterodimers aligned head-to-tail. Here we have analyzed GTP exchange and GTP hydrolysis at the exchangeable GTP-binding site (E-site) of tubulin heterodimers in complex with Op18, RB3, or SCG10. These proteins stimulate a low and indistinguishable rate of GTP hydrolysis, and our results show that GTP exchange is blocked at both E-sites of the ternary complex, whereas GTP hydrolysis only occurs at one of the two E-sites. Results from mutational analysis of clusters of hydrophobic residues within the first helical repeat of Op18 suggest that GTP is hydrolyzed at the E-site that is interfaced between the head-to-tail arranged heterodimers, which is consistent with predicted GTPase productive interactions between the two tubulin heterodimers. Our mutational analysis has also indicated that Op18/stathmin family members actively restrain the otherwise potent GTPase productive interactions that are generated by longitudinal interactions within protofilaments. We conclude that tubulin heterodimers in complex with Op18/stathmin family members are subject to allosteric effects that prevent futile cycles of GTP hydrolysis.