Cytokinesis mediated through the recruitment of cortexillins into the cleavage furrow.

The fact that substrate-anchored Dictyostelium cells undergo cytokinesis in the absence of myosin II underscores the importance of other proteins in enabling the cleavage furrow to constrict. Cortexillins, a pair of actin-bundling proteins, are required for normal cleavage. They are targeted to the incipient furrow in wild-type and, more prominently, in myosin II-null cells. No other F-actin bundling or cross-linking protein tested is co-localized. Green fluorescent protein fusions show that the N-terminal actin-binding domain of cortexillin I is dispensable and the C-terminal region is sufficient for translocation to the furrow and the rescue of cytokinesis. Cortexillins are suggested to have a targeting signal for coupling to a myosin II-independent system that directs transport of membrane proteins to the cleavage furrow.     

Chlamydia trachomatis inclusions induce asymmetric cleavage furrow formation and ingression failure in host cells

Chlamydia trachomatis infection has been suggested to induce host genome duplication and is linked to increased risks of cervical cancer. We describe here the mechanism by which Chlamydia causes a cleavage furrow defect that consistently results in the formation of multinucleated host cells, a phenomenon linked to tumorigenesis. Host signaling proteins essential for cleavage furrow initiation, ingression, and stabilization are displaced from one of the prospective furrowing cortices after Chlamydia infection. This protein displacement leads to the formation of a unique asymmetrical, unilateral cleavage furrow in infected human cells. The asymmetrical distribution of signaling proteins is caused by the physical presence of the Chlamydia inclusion at the cell equator. By using ingested latex beads, we demonstrate that the presence of a large vacuole at the cell equator is sufficient to cause furrow ingression failure and can lead to multinucleation. Interestingly, internalized latex beads of similar size do not localize to the cell equator as efficiently as Chlamydia inclusions; moreover, inhibition of bacterial protein synthesis with antibiotic reduces the frequency at which Chlamydia localizes to the cell equator. Together, these results suggest that Chlamydia effectors are involved in strategic positioning of the inclusion during cell division.     

Cytokinesis and cytochalasin-induced furrow regression in the first-cleavage zygote of Xenopus laevis

Summary The egg cleavage and the cytochalasin effect has been investigated in the first-cleavage zygotes of Xenopus laevis.—Furrow formation results from the joint action of surface constriction, junction formation, and ingrowth of new membrane. During the constriction phase lanthanum-binding exudate is deposited in the furrow gap. This material is distributed in dispersed patches (Ø 200 Å) giving rise to a bur surface which coats interdigitating cell protrusions. At places where protrusions meet they form 160 Å wide adherent junctions which provisionally fix the contracted furrow. At the end of the constriction phase (which ultimately accounts for 15 per cent of the reduction in egg diameter in the plane of cleavage) the layer of 100 Å filaments beneath the furrow bottom is split by local ingrowth of new membrane, and the filaments take up lateral positions. Furrow ingrowth proceeds by bilateral insertion of new membrane.The application of 7.5 g/ml cytochalasin B (CCB) leads to furrow regression without blocking contractility. CCB primarily affects the cell surface, and only indirectly affects the microfilament system. It interferes with cell junction formation and deranges furrow ingrowth. In the absence of stable 160 Å wide, adherent junctions the new membrane grows outwards instead of inwards. The results are discussed with reference to furrow regression induced by other membrane-destabilizing agents such as phospholipase C. Comparison reveals that CCB in addition facilitates the insertion of new cell membrane—. To interpret the biological effects of cytochalasin an alternative working hypothesis is presented, which meets the objections that can be raised against the concept that cytochalasin B specifically interferes with thin microfilaments.Dedicated with deep respect to Prof. Dr. Chr. P. Raven at the occasion of his 65th birthday.I thank Dr. S. B. Carter for a supply of cytochalasin B. I am grateful to my fellow-staff members of the Hubrecht Laboratory, and in particular to Prof. P. D. Nieuwkoop, for constructive criticism and valuable suggestions. I should like to thank Mr. E. van Voorst for his technical assistance, and Miss Eva Bartová, Mr. L. Boom and Mr. R. Tokaya for preparing the prints and the drawing. I am also indebted to Dr. J. Faber for editorial assistance, and to Drs. P. H. Ververgaert for his help in carrying out the densitometric measurements.     

Recruitment of cortexillin into the cleavage furrow is controlled by Rac1 and IQGAP-related proteins.

Cytokinesis in eukaryotic organisms is under the control of small GTP-binding proteins, although the underlying molecular mechanisms are not fully understood. Cortexillins are actin-binding proteins whose activity is crucial for cytokinesis in Dictyostelium. Here we show that the IQGAP-related and Rac1-binding protein DGAP1 specifically interacts with the C-terminal, actin-bundling domain of cortexillin I. Like cortexillin I, DGAP1 is enriched in the cortex of interphase cells and translocates to the cleavage furrow during cytokinesis. The activated form of the small GTPase Rac1A recruits DGAP1 into a quaternary complex with cortexillin I and II. In DGAP1(-) mutants, a complex can still be formed with a second IQGAP-related protein, GAPA. The simultaneous elimination of DGAP1 and GAPA, however, prevents complex formation and localization of the cortexillins to the cleavage furrow. This leads to a severe defect in cytokinesis, which is similar to that found in cortexillin I/II double-null mutants. Our observations define a novel and functionally significant signaling pathway that is required for cytokinesis.     

A barrier to lateral diffusion in the cleavage furrow of dividing mammalian cells

Barriers to diffusion of proteins and lipids play an important role in generating functionally specialized regions of the plasma membrane. Such barriers have been reported at the base of axons, at the bud neck in Saccharomyces cerevisiae, as well as at the tight junctions of epithelia. How diffusion barriers are formed and how they effect behavior of both inner and outer leaflets of the bilayer are not fully understood. Here, we provide evidence for a cortical barrier to diffusion within the cleavage furrow of mammalian cells. Photobleaching-based assays were used to measure diffusion of three membrane proteins with differing topologies and putative lipid raft association, as well as the lipid analog dialkylindocarbocyanine (DiI C18, ), across the cleavage furrow. There was a block in diffusion of proteins with a cytosolic domain, but not of proteins anchored in the outer leaflet of the PM or of DiI. Diffusion of lipid raft proteins in the inner and outer leaflets of the membrane was not directly coupled. The distribution of Septin proteins, as opposed to cortical actin, was consistent with a functional role in limiting diffusion.     

Alpha-actinin localization in the cleavage furrow during cytokinesis

We used antibodies against alpha-actinin and myosin labeled directly with contrasting fluorochromes to localize these contractile proteins simultaneously in dividing chick embryo cells. During mitosis anti-alpha-actinin stains diffusely the entire cytoplasm including the mitotic spindle, while in the same cells intense antimyosin staining delineates the spindle. During cytokinesis both antibodies stain the cleavage furrow intensely, and until the midbody forms the two staining patterns in the same cell are identical at the resolution of the light microscope. Thereafter the anti-alpha-actinin staining of the furrow remains strong, but the antimyosin staining diminishes. These observations suggest that alpha-actinin participates along with actin and myosin in the membrane movements associated with cytokinesis.     

Stress fiber and cleavage furrow formation in living cells microinjected with fluorescently labeled alpha-actinin

alpha-Actinins, isolated from muscle and nonmuscle sources and labeled with various fluorescent dyes, were microinjected into living PtK2 cells during interphase to observe the reformation of stress fibers following cell division. Fluorescently labeled ovalbumin and bovine serum albumin were also injected as control proteins. alpha-Actinin was incorporated into stress fibers within 5 minutes after injection and remained present in the fibers for up to 11 days. The pattern of incorporation was the same regardless of whether the alpha-actinin was isolated from muscle or nonmuscle tissues or whether it was labeled with fluorescein, Lucifer Yellow, or rhodamine dyes. In contrast, neither labeled ovalbumin nor bovine serum albumin were incorporated into stress fibers. When the injected cells entered prophase, all stress fibers disassembled, resulting in a distribution of the fluorescent alpha-actinin throughout the cytoplasm. During cytokinesis, the fluorescent alpha-actinin was concentrated in the broad area between the separated chromosomes and along the edge of the cell in the cleavage area. Within 10 minutes after the completion of cleavage, the first fluorescent stress fibers reformed parallel to the spreading edges of the daughter cells and in close association with the midbody with a concomitant loss of alpha-actinin in the former cleavage furrow. Additional fibers formed adjacent to these first stress fibers. In some cases, new stress fibers formed between two existing stress fibers and some stress fibers moved up to 4 micron apart from one another in the course of 2 hours. Thus, fluorescent alpha-actinin, injected into living cells, undergoes the same cyclical changes in distribution as endogenous alpha-actinin during the cell cycle: from stress fibers to cleavage furrow and back to stress fibers.     

Two-step positioning of a cleavage furrow by cortexillin and myosin II

BACKGROUND: Myosin II, a conventional myosin, is dispensable for mitotic division in Dictyostelium if the cells are attached to a substrate, but is required when the cells are growing in suspension. Only a small fraction of myosin II-null cells fail to divide when attached to a substrate. Cortexillins are actin-bundling proteins that translocate to the midzone of mitotic cells and are important for the formation of a cleavage furrow, even in attached cells. Here, we investigated how myosin II and cortexillin I cooperate to determine the position of a cleavage furrow. RESULTS: Using a green fluorescent protein (GFP)-cortexillin I fusion protein as a marker for priming of a cleavage furrow, we found that positioning of a cleavage furrow occurred in two steps. In the first step, which was independent of myosin II and substrate, cortexillin I delineated a zone around the equatorial region of the cell. Myosin II then focused the cleavage furrow to the middle of this cortexillin I zone. If asymmetric cleavage in the absence of myosin II partitioned a cell into a binucleate and an anucleate portion, cell-surface ruffles were induced along the cleavage furrow, which led to movement of the anucleate portion along the connecting strand towards the binucleate one. CONCLUSIONS: In myosin II-null cells, cleavage furrow positioning occurs in two steps: priming of the furrow region and actual cleavage, which may proceed in the middle or at one border of the cortexillin ring. A control mechanism acting at late cytokinesis prevents cell division into an anucleate and a binucleate portion, causing a displaced furrow to regress if it becomes aberrantly located on top of polar microtubule asters.     

On the mechanism of cleavage furrow ingression in Dictyostelium

The ability of Dictyostelium cells to divide without myosin II in a cell cycle-coupled manner has opened two questions about the mechanism of cleavage furrow ingression. First, are there other possible functions for myosin II in this process except for generating contraction of the furrow by a sliding filament mechanism? Second, what could be an alternative mechanical basis for the furrowing? Using aberrant changes of the cell shape and anomalous localization of the actin-binding protein cortexillin I during asymmetric cytokinesis in myosin II-deficient cells as clues, it is proposed that myosin II filaments act as a mechanical lens in cytokinesis. The mechanical lens serves to focus the forces that induce the furrowing to the center of the midzone, a cortical region where cortexillins are enriched in dividing cells. Additionally, continual disassembly of a filamentous actin meshwork at the midzone is a prerequisite for normal ingression of the cleavage furrow and a successful cytokinesis. If this process is interrupted, as it occurs in cells that lack cortexillins, an overassembly of filamentous actin at the midzone obstructs the normal cleavage. Disassembly of the crosslinked actin network can generate entropic contractile forces in the cortex, and may be considered as an alternative mechanism for driving ingression of the cleavage furrow. Instead of invoking different types of cytokinesis that operate under attached and unattached conditions in Dictyostelium, it is anticipated that these cells use a universal multifaceted mechanism to divide, which is only moderately sensitive to elimination of its constituent mechanical processes.     

Concentration of caveolin-1 in the cleavage furrow as revealed by time-lapse analysis

Caveolin-1 is a major component of caveolae. Recent studies have suggested a possible role of caveolin-1 in cell transformation and normal cell proliferation. To observe the behavior of caveolin-1 in living mitotic cells, we prepared cDNA constructs encoding the chimeric protein of alpha- or beta-caveolin-1 and green fluorescent protein (GFP) and transfected culture cells with them. Correct targeting of the chimera to the caveolae was confirmed by colocalization with the caveolar inositol 1,4,5-trisphosphate receptor-like protein. By time-lapse observation of mitotic MDCKII cells, the GFP-caveolin-1 chimeras were seen throughout the plasma membrane before cell division, but became markedly concentrated at the cleavage furrow during cytokinesis. Accumulation around the spindle poles was also observed at late telophase. The result showed that caveolin-1 undergoes a drastic distributional change during cell division and suggested that the protein may be involved in the cytokinetic process.     

Both daughter cells traffic and exocytose membrane at the cleavage furrow during mammalian cytokinesis

Membrane trafficking during cytokinesis is not well understood. We used advanced live cell imaging techniques to track exocytosis of single vesicles to determine whether constitutively exocytosed membrane is focally delivered to the cleavage furrow. Ultrasensitive three-dimensional confocal time-lapse imaging of the temperature-sensitive membrane cargo protein vesicular stomatitis virus protein-yellow fluorescent protein revealed that vesicles from both daughter cells traffic out of the Golgi and into the furrow, following curvilinear paths. Immunolocalization and photobleaching experiments indicate that individual vesicles accumulate at the midbody and generate a reserve vesicle pool that is distinct from endosomal and lysosomal compartments. Total internal reflection fluorescence microscopy imaging provided direct evidence that Golgi-derived vesicles from both daughter cells not only traffic to the furrow region but dock and fuse there, supporting a symmetrically polarized exocytic delivery model. In contrast, quantitative analysis of midbody abscission showed inheritance of the midbody remnant by one daughter cell, indicating that cytokinesis is composed of both symmetrical and asymmetrical stages.     

p190RhoGAP negatively regulates Rho activity at the cleavage furrow of mitotic cells

Previous studies demonstrated that p190RhoGAP (p190) negatively affects cytokinesis in a RhoGAP-dependent manner, suggesting that regulation of Rho may be a critical mechanism of p190 action during cytokinesis. P190 localizes to the cleavage furrow (CF) of dividing cells, and its levels decrease during late mitosis by an ubiquitin-mediated mechanism, consistent with the hypothesis that high RhoGTP levels are required for completion of cytokinesis. To determine whether RhoGTP levels in the CF are affected by p190 and to define the phase(s) of cytokinesis in which p190 is involved, we used FRET analysis alone or in combination with time-lapse microscopy. In normal cell division activated Rho accumulated at the cell equator in early anaphase and in the contractile ring, where it co-localized with p190. Real-time movies revealed that cells expressing elevated levels of p190 exhibited multiple cycles of abnormal CF site selection and ingression/regression, which resulted in failed or prolonged cytokinesis. This was accompanied by mislocalization of active Rho at the aberrant CF sites. Quantified data revealed that in contrast to ECT2 and dominate negative p190 (Y1283Ap190), which resulted in hyper-activated Rho, Rho activity in the CF was reduced by wild type p190 in a dose-dependent manner. These results suggest that p190 regulates cytokinesis through modulation of RhoGTP levels, thereby affecting CF specification site selection and subsequent ring contraction.     

Asymmetric cortical extension shifts cleavage furrow position in Drosophila neuroblasts

The cytokinetic cleavage furrow is typically positioned symmetrically relative to the cortical cell boundaries, but it can also be asymmetric. The mechanisms that control furrow site specification have been intensively studied, but how polar cortex movements influence ultimate furrow position remains poorly understood. We measured the position of the apical and the basal cortex in asymmetrically dividing Drosophila neuroblasts and observed preferential displacement of the apical cortex that becomes the larger daughter cell during anaphase, effectively shifting the cleavage furrow toward the smaller daughter cell. Asymmetric cortical extension is correlated with the presence of cortical myosin II, which is polarized in neuroblasts. Loss of myosin II asymmetry by perturbing heterotrimeric G-protein signaling results in symmetric extension and equal-sized daughter cells. We propose a model in which contraction-driven asymmetric polar extension of the neuroblast cortex during anaphase contributes to asymmetric furrow position and daughter cell size.     

Loss of function in phenylketonuria is caused by impaired molecular motions and conformational instability

A significant share of patients with phenylalanine hydroxylase (PAH) deficiency benefits from pharmacological doses of tetrahydrobiopterin (BH(4)), the natural PAH cofactor. Phenylketonuria (PKU) is hypothesized to be a conformational disease, with loss of function due to protein destabilization, and the restoration of enzyme function that is observed in BH(4) treatment might be transmitted by correction of protein misfolding. To elucidate the molecular basis of functional impairment in PAH deficiency, we investigated the impact of ten PAH gene mutations identified in patients with BH(4)-responsiveness on enzyme kinetics, stability, and conformation of the protein (F55L, I65S, H170Q, P275L, A300S, S310Y, P314S, R408W, Y414C, Y417H). Residual enzyme activity was generally high, but allostery was disturbed in almost all cases and pointed to altered protein conformation. This was confirmed by reduced proteolytic stability, impaired tetramer assembly or aggregation, increased hydrophobicity, and accelerated thermal unfolding--with particular impact on the regulatory domain--observed in most variants. Three-dimensional modeling revealed the involvement of functionally relevant amino acid networks that may communicate misfolding throughout the protein. Our results substantiate the view that PAH deficiency is a protein-misfolding disease in which global conformational changes hinder molecular motions essential for physiological enzyme function. Thus, PKU has evolved from a model of a genetic disease that leads to severe neurological impairment to a model of a treatable protein-folding disease with loss of function.     

Chromosome instability in colorectal tumor cells is associated with defects in microtubule plus-end attachments caused by a dominant mutation in APC

The attachment of microtubule plus ends to kinetochores and to the cell cortex is essential for the fidelity of chromosome segregation. Here, we characterize the causes underlying the high rates of chromosome instability (CIN+) observed in colorectal tumor cells. We show that CIN+ tumor cells exhibit inefficient microtubule plus-end attachments during mitosis, accompanied by impairment of chromosome alignment in metaphase. The mitotic abnormalities associated with CIN+ tumor cells correlated with status of adenomatous polyposis coli (APC). Importantly, we have shown that a single truncating mutation in APC, similar to mutations found in tumor cells, acts dominantly to interfere with microtubule plus-end attachments and to cause a dramatic increase in mitotic abnormalities. We propose that APC functions to modulate microtubule plus-end attachments during mitosis, and that a single mutant APC allele predisposes cells to increased mitotic abnormalities, which may contribute to tumor progression.