Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure.

At cell division, the mammalian kinetochore binds many spindle microtubules that make up the kinetochore-fiber. To segregate chromosomes, the kinetochore-fiber must be dynamic and generate and respond to force. Yet, how it remodels under force remains poorly understood. Kinetochore-fibers cannot be reconstituted in vitro, and exerting controlled forces in vivo remains challenging. Here, we use microneedles to pull on mammalian kinetochore-fibers and probe how sustained force regulates their dynamics and structure. We show that force lengthens kinetochore-fibers by persistently favoring plus-end polymerization, not by increasing polymerization rate. We demonstrate that force suppresses depolymerization at both plus and minus ends, rather than sliding microtubules within the kinetochore-fiber. Finally, we observe that kinetochore-fibers break but do not detach from kinetochores or poles. Together, this work suggests an engineering principle for spindle structural homeostasis: different physical mechanisms of local force dissipation by the k-fiber limit force transmission to preserve robust spindle structure. These findings may inform how other dynamic, force-generating cellular machines achieve mechanical robustness.

Body mass-specific Na+-K+-ATPase activity in the medullary thick ascending limb: implications for species-dependent urine concentrating mechanisms.

In general, the mammalian whole body mass-specific metabolic rate correlates positively with maximal urine concentration (Umax) irrespective of whether or not the species have adapted to arid or mesic habitat. Accordingly, we hypothesized that the thick ascending limb (TAL) of a rodent with markedly higher whole body mass-specific metabolism than rat exhibits a substantially higher TAL metabolic rate as estimated by Na+-K+-ATPase activity and Na+-K+-ATPase α1-gene and protein expression. The kangaroo rat inner stripe of the outer medulla exhibits significantly higher mean Na+-K+-ATPase activity (~70%) compared with two rat strains (Sprague-Dawley and Munich-Wistar), extending prior studies showing rat activity exceeds rabbit. Furthermore, higher expression of Na+-K+-ATPase α1-protein (~4- to 6-fold) and mRNA (~13-fold) and higher TAL mitochondrial volume density (~20%) occur in the kangaroo rat compared with both rat strains. Rat TAL Na+-K+-ATPase α1-protein expression is relatively unaffected by body hydration status or, shown previously, by dietary Na+, arguing against confounding effects from two unavoidably dissimilar diets: grain-based diet without water (kangaroo rat) or grain-based diet with water (rat). We conclude that higher TAL Na+-K+-ATPase activity contributes to relationships between whole body mass-specific metabolic rate and high Umax. More vigorous TAL Na+-K+-ATPase activity in kangaroo rat than rat may contribute to its steeper Na+ and urea axial concentration gradients, adding support to a revised model of the urine concentrating mechanism, which hypothesizes a leading role for vigorous active transport of NaCl, rather than countercurrent multiplication, in generating the outer medullary axial osmotic gradient.

Protein kinase A governs a RhoA-RhoGDI protrusion-retraction pacemaker in migrating cells.

The cyclical protrusion and retraction of the leading edge is a hallmark of many migrating cells involved in processes such as development, inflammation and tumorigenesis. The molecular identity of the signalling mechanisms that control these cycles has remained unknown. Here, we used live-cell imaging of biosensors to monitor spontaneous morphodynamic and signalling activities, and employed correlative image analysis to examine the role of cyclic-AMP-activated protein kinase A (PKA) in protrusion regulation. PKA activity at the leading edge is closely synchronized with rapid protrusion and with the activity of RhoA. Ensuing PKA phosphorylation of RhoA and the resulting increased interaction between RhoA and RhoGDI (Rho GDP-dissociation inhibitor) establish a negative feedback mechanism that controls the cycling of RhoA activity at the leading edge. Thus, cooperation between PKA, RhoA and RhoGDI forms a pacemaker that governs the morphodynamic behaviour of migrating cells.

Requirement for formin-induced actin polymerization during spread of Shigella flexneri.

Actin polymerization in the cytosol and at the plasma membrane is locally regulated by actin nucleators. Several microbial pathogens exploit cellular actin polymerization to spread through tissue. The movement of the enteric pathogen Shigella flexneri, both within the cell body and from cell to cell, depends on actin polymerization. During intercellular spread, actin polymerization at the bacterial surface generates protrusions of the plasma membrane, which are engulfed by adjacent cells. In the cell body, polymerization of actin by Shigella spp. is dependent on N-WASP activation of the Arp2/Arp3 complex. Here we demonstrate that, in contrast, efficient protrusion formation and intercellular spread depend on actin polymerization that involves activation of the Diaphanous formin Dia. While the Shigella virulence protein IpgB2 can bind and activate Dia1 (N. M. Alto et al., Cell 124:133-145, 2006), its absence does not result in a detectable defect in Dia-dependent protrusion formation or spread. The dependence on the activation of Dia during S. flexneri infection contrasts with the inhibition of this pathway observed during vaccinia virus infection.

Phosphoinositide 3-kinase is required for intracellular Listeria monocytogenes actin-based motility and filopod formation.

Motile nonmuscle cells concentrate phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in areas of new actin filament assembly. There is great interest in assessing the in vivo functional significance of these phosphoinositides, and we have used Listeria monocytogenes to explore the contribution of PtdIns(3,4,5)P3 and PtdIns(4,5)P2 to its actin-based motility. In Listeria-infected PtK2 cells Akt-pleckstrin homology (PH)-green fluorescent protein (GFP) and phospholipase C delta (PLC delta)-PH-GFP both first concentrate at the front of motile Listeria, subsequently surrounding the bacterium and then concentrating in the actin filament tail. Surprisingly, Listeria ActA mutant strains lacking the putative phosphoinositide binding site are also able to concentrate these probes. Reduction of available PtdIns(3,4,5)P3 by expression of Akt-PH-GFP and available PtdIns(4,5)P2 by expression of PLC delta-PH-GFP both significantly slow Listeria actin-based movement. Treatment of cells with the PI 3-kinase inhibitor, LY294002, dissociates Akt-PH but not PLC delta-PH, from the bacterial surface and cell membranes, and results in near complete inhibition of Listeria actin-based motility and filopod formation. Removal of LY294002 results in rapid and full recovery of Akt-PH localization, Listeria actin-based motility, and filopod formation. These findings suggest that PtdIns(4,5)P2 is concentrated at the surface of Listeria and serves as the substrate for PtdIns(3,4,5)P3 production, indicating a central role for PI 3-kinases in Listeria intracellular actin-based motility and filopod formation.

Membrane organization and function of M1 and M23 isoforms of aquaporin-4 in epithelial cells.

Aquaporin-4 (AQP4) water channels exist as heterotetramers of M1 and M23 splice variants and appear to be present in orthogonal arrays of intramembraneous particles (OAPs) visualized by freeze-fracture microscopy. We report that AQP4 forms OAPs in rat gastric parietal cells but not in parietal cells from the mouse or kangaroo rat. Furthermore, the organization of principal cell OAPs in Brattleboro rat kidney is perturbed by vasopressin (arginine vasopressin). Membranes of LLC-PK(1) cells expressing M23-AQP4 showed large, abundant OAPs, but none were detectable in cells expressing M1-AQP4. Measurements of osmotic swelling of transfected LLC-PK(1) cells using videomicroscopy, gave osmotic water permeability coefficient (P(f)) values (in cm/s) of 0.018 (M1-AQP4), 0.019 (M23-AQP4), and 0.003 (control). Quantitative immunoblot and immunofluorescence showed an eightfold greater expression of M1- over M23-AQP4 in the cell lines, suggesting that single-channel p(f) (cm(3)/s) is much greater for the M23 variant. Somatic fusion of M1- and M23-AQP4 cells (P(f) = 0.028 cm/s) yielded OAPs that were fewer and smaller than in M23 cells alone, and M1-to-M23 expression ratios ( approximately 1:4) normalized to AQP4 in M1 or M23 cells indicated a reduced single-channel p(f) for the M23 variant. Expression of an M23-AQP4-Ser(111E) mutant produced approximately 1.5-fold greater single-channel p(f) and OAPs that were up to 2.5-fold larger than wild-type M23-AQP4 OAPs, suggesting that a putative PKA phosphorylation site Ser(111) is involved in OAP formation. We conclude that the higher-order organization of AQP4 in OAPs increases single-channel osmotic water permeability by one order of magnitude and that differential cellular expression levels of the two isoforms could regulate this organization.

Partitioning of aquaporin-4 water channel mRNA and protein in gastric glands.

Immunolocalization studies in proximal, middle, and distal stomach indicated that aquaporin-4 (AQP4) protein is localized only in parietal cells located in the middle or deep regions of the gastric glands. In studies using in situ hybridization, AQP4 mRNA failed to localize in parietal cells but was identified in neighboring mucosal cells that were triangular in shape and smaller than parietal cells in size, and in columnar cells at the base of the gastric gland. This spatial separation of mRNA and protein was also observed in other species and with other kind of mRNA/protein. In neonatal and adolescent rats, the appearance of morphologically mature parietal cells was preceded by identification of mRNA-bearing triangular cells. Cells harboring both protein and mRNA were observed in postnatal rats and in the pyloric region of the glandular stomach, during induced hypergastrinaemia. The results suggest that such cells represent a transition between those that bear only mRNA and those that are terminally differentiated, expressing proteins that are related to acid secretion.

[Effect of laminin on numerical karyotype variability of kangaroo rat kidney cell lines]. / Vliianie laminina na kolichestvennuiu kariotipicheskuiu izmenchivost' v kletochnykh liniiakh pochki kengurovoi krysy.

The numerical karyotypic variability has been investigated in "markerless" epithelial-like Rat kangaroo kidney cell lines NBL-3-11 and NBL-3-17 on cultivation on a laminin-2/4 coated surface. In cell line NBL-3-17, cultivated on the laminin-coated surface for 2, 4 and 12 days, the character of numerical karyotypic variability has changed. In 2 days the general character of cell distribution for the chromosome number did not change, but the frequency of cells with modal number of chromosomes decreases significantly, while that of cells with lower chromosome number show a tendency to increase. At a prolongation of cultivation time to 4 and 12 days, the numerical karyotypic heterogeneity in cell population increases due to a significant change in the general character of cell distribution for the chromosome number, which is caused by a significant decrease in the frequency of cells with the modal number of chromosomes, and by an increase in the frequency of cells with lower chromosome number. The analysis of distribution of individual chromosomes showed that the number of types of additional structural variants of the karyotype (SVK) increases significantly on cultivation on laminin for 2-12 days. In cell line NBL-3-11, cultivated on the laminin-coated surface for 2 and 4 days, the character of numerical karyotypic variability did not change compared to control variants. Possible reasons of the observed changes of numerical karyotypic variability in cell line NBL-3-17 is discussed. The reason of differences in the character of numerical karyotypic variability between cell lines NBL-3-11 and NBL-3-17 possibly consists in the change of gene expression, namely in a dose of certain functioning genes. The polymerase chain reaction with arbitrary primers revealed no differences between DNA patterns of cell lines NBL-3-17 and NBL-3-11. This can reflect a similarity in the primary DNA structure of both cell lines. Hence, these lines differ only in the number of homologous chromosomes (hypotriploid and hypodiploid).

[Effect of laminin on structural karyotype variability of kangaroo rat kidney cell lines]. / Vliianie laminina na strukturnuiu kariotipicheskuiu izmenchivnost' v kletochnykh liniiakh pochki kengurovoi krysy.

The structural karyotypic variability has been investigated in the "markerless" epithelial-like Rat kangaroo kidney cell lines NBL-3-17 and NBL-3-11 on cultivation on a laminin-2/4 coated surface. In cell line NBL-3-17, cultivated on the laminin-coated surface for 2, 4 and 12 days, and in cell line NBL-3-11, cultivated on the laminin-coated surface for 2 and 4 days, there is a significant increase in the frequency of chromosomal aberrations, both chromosomal breaks and dicentrics (telomeric associations). Different sensitivity of individual chromosomes to inducing chromosomal breaks was observed in addition to a preferential involvement of some chromosomes in dicentric formation. Structural instability of chromosomes at cultivation on laminin demonstrates nonspecific reaction of the "markerless" cell lines to unfavourable factors of the environment. We discuss possible reasons of differences in the character of karyotypic variability between a cell line of the Indian muntjac skin fibroblasts and epithelial-like Rat kangaroo kidney cell lines cultivated on laminin.

Investigation of the effects of extracts of Manilkara zapota (L.) P. van Royen on blood pressure and heart rate of rats

The objective of the study was to investigate the hypotensive effect produced by leaf extracts of Manilkara zapota. Methanol extracts of Manilkara zapota leaves were prepared using a soxhlet apparatus. The methanol was removed with a rotor evaporator. Sprague-Dawley rats were anaesthetized with urethane (1.2 mg/kb) and doses of 0.63, 1.25, 2.5, 5.0, 10.0 and 20.0 mg/kg body weight of extract were administered intavenously. Saline (0.9 percent) was given as a control. The effects on blood pressure and heart rate were recorded using a Pressure transducer (Spectramed model 23XL) coupled to a Grass polygraph (model 79E). The plant extract showed a dose-related hypotensive activity and no significant change in heart rate (P.0.05). Toxicity was observed with doses greater than 20mg/kg body weight. These results indicate that Manilkara zapota leaf tea used in folklore medicine to treat hypertension does indeed show various degrees of hypotensive activity when tested in lab animals. However, larger doses were toxic. It is therefore necessary to assess the beneficial as well as the adverse effects of this herb before usage by the hypertensive patient

Both alpha and beta isoforms of mammalian DNA topoisomerase II associate with chromosomes in mitosis.

Two isoforms of DNA topoisomerase II, alpha and beta, coded by separate genes, are expressed in actively cycling vertebrate cells. Some previous studies have suggested that only topoisomerase II alpha remains associated with chromosomes at mitosis. Here, the distributions of topoisomerase II alpha and beta in mitosis were studied by subcellular fractionation and by immunolocalization. Both isoforms of topoisomerase II were found to remain associated with mitotic chromatin. Topoisomerase II alpha was distributed along chromosome arms throughout mitosis and was highly concentrated at centromeres until mid-anaphase, particularly in some cell types. Topoisomerase II beta showed weak concentration at centromeres in early mitosis in some cell types and was distributed along chromosome arms at every stage of mitosis through telophase. These studies suggest that in most cells both the major topoisomerase II isoforms may play roles in chromatin remodeling during M phase.

A fluorescent resonant energy transfer-based biosensor reveals transient and regional myosin light chain kinase activation in lamella and cleavage furrows.

Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases.

Dolastatin 11, a marine depsipeptide, arrests cells at cytokinesis and induces hyperpolymerization of purified actin.

The successful synthesis of dolastatin 11, a depsipeptide originally isolated from the mollusk Dolabella auricularia, permitted us to study its effects on cells. The compound arrested cells at cytokinesis by causing a rapid and massive rearrangement of the cellular actin filament network. In a dose-and time-dependent manner, F-actin was rearranged into aggregates, and subsequently the cells displayed dramatic cytoplasmic retraction. The effects of dolastatin 11 were most similar to those of the sponge-derived depsipeptide jasplakinolide, but dolastatin 11 was about 3-fold more cytotoxic than jasplakinolide in the cells studied. Like jasplakinolide, dolastatin 11 induced the hyperassembly of purified actin into filaments of apparently normal morphology. Dolastatin 11 was qualitatively more active than jasplakinolide and, in a quantitative assay we developed, dolastatin 11 was twice as active as jasplakinolide and 4-fold more active than phalloidin. However, in contrast to jasplakinolide and phalloidin, dolastatin 11 did not inhibit the binding of a fluorescent phalloidin derivative to actin polymer nor was it able to displace the phalloidin derivative from polymer. Thus, despite its structural similarity to other agents that induce actin assembly (all are peptides or depsipeptides), dolastatin 11 may interact with actin polymers at a distinct drug binding site.

Single amino acid (arginine) deprivation: rapid and selective death of cultured transformed and malignant cells.

The effects of arginine deprivation (-Arg) has been examined in 26 cell lines. Less than 10% of those with transformed or malignant phenotype survived for > 5 days, and many died more rapidly, notably leukaemic cells. Bivariate flow cytometry confirmed that vulnerable cell lines failed to move out of cell cycle into a quiescent state (G0), but reinitiated DNA synthesis. Many cells remained in S-phase, and/or had difficulty progressing through to G2 and M. Two tumour lines proved relatively 'resistant', A549 and MCF7. Although considerable cell loss occurred initially, both lines showed a 'cell cycle freeze', in which cells survived for > 10 days. These cells recovered their proliferative activity in +Arg medium, but behaved in the same manner to a second -Arg episode as they did to the first episode. In contrast, normal cells entered G0 and survived in -Arg medium for several weeks, with the majority of cells recovering with predictable kinetics in +Arg medium. In general, cells from a wide range of tumours and established lines die quickly in vitro following -Arg treatment, because of defective cell cycle checkpoint stringency, the efficacy of the treatment being most clearly demonstrated in co-cultures in which only the normal cells survived. The findings demonstrate a potentially simple, effective and non-genotoxic strategy for the treatment of a wide range of cancers.

Temporal and spatial control of cyclin B1 destruction in metaphase.

The proteolysis of key regulatory proteins is thought to control progress through mitosis. Here we analyse cyclin B1 degradation in real time and find that it begins as soon as the last chromosome aligns on the metaphase plate, just after the spindle-assembly checkpoint is inactivated. At this point, cyclin B1 staining disappears from the spindle poles and from the chromosomes. Cyclin B1 destruction can subsequently be inactivated throughout metaphase if the spindle checkpoint is reimposed, and this correlates with the reappearance of cyclin B1 on the spindle poles and the chromosomes. These results provide a temporal and spatial link between the spindle-assembly checkpoint and ubiquitin-mediated proteolysis.

Regional regulation of microtubule dynamics in polarized, motile cells.

Microtubules are known to be required for locomotion of mammalian cells, and recent experiments demonstrate that suppression of microtubule dynamic turnover reduces the rate of cell motility and induces wandering of growth cones [Liao et al., 1995: J Cell Sci. 108:3473-3483; Tanaka et al., 1995: J Cell Biol. 128:139-155]. To determine how microtubule dynamic instability behavior contributes to directed cell locomotion, the behavior of individual microtubules has been directly observed and quantified at leading and lateral edges of hepatocyte growth factor-treated motile cells. Microtubules extended into newly formed protrusions at the leading edge; these "pioneer" microtubules [Waterman-Storer and Salmon, 1997: J Cell Biol. 139:417-434] showed persistent growth when compared with microtubules in non-leading, lateral edges. The percentage of total observation time spent in the growth phase was 68.2% at the leading edge compared with 32.0% in non-leading edges, and net microtubule elongation was observed in lamellipodia at the leading edge. The frequency of catastrophe transitions was threefold greater and the average number of transitions/microtubule/min was twofold greater in non-leading edges, as compared with the leading edge. These observations demonstrate that pioneer microtubules that enter newly formed lamellipodia at the leading edge of motile cells are characterized by persistent growth excursions, and directly demonstrate that the frequency of catastrophe transitions can be regionally regulated in polarized motile cells. The data indicate that region specific differences in the organization and dynamics of actin filaments may regulate microtubule dynamic instability behavior in vivo.

Characterization of the p22 subunit of dynactin reveals the localization of cytoplasmic dynein and dynactin to the midbody of dividing cells.

Dynactin, a multisubunit complex that binds to the microtubule motor cytoplasmic dynein, may provide a link between dynein and its cargo. Many subunits of dynactin have been characterized, elucidating the multifunctional nature of this complex. Using a dynein affinity column, p22, the smallest dynactin subunit, was isolated and microsequenced. The peptide sequences were used to clone a full-length human cDNA. Database searches with the predicted amino acid sequence of p22 indicate that this polypeptide is novel. We have characterized p22 as an integral component of dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments indicate that p22 binds directly to the p150(Glued) subunit of dynactin. Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis. Antibodies to p22, as well as to other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing cells; cytoplasmic dynein was also localized to these structures. We therefore propose that dynein/dynactin complexes may have a novel function during cytokinesis.

Subcellular phototoxicity of 5-aminolaevulinic acid (ALA).

BACKGROUND AND OBJECTIVE: 5-aminolaevulinic acid (ALA) is a new, promising photosensitizer for PDT of cancer. Subcellular toxicity induced by ALA and light exposure in single cells was studied to elucidate the mechanism of cell damage. STUDY DESIGN/MATERIALS AND METHODS: CPAE, PTK2, and rat neonatal myocardial cells treated with ALA were examined for localization using fluorescence microscopy and for subcellular phototoxicity using 630 nm laser microbeam irradiation of specific subcellular regions. RESULTS: In CPAE and PTK2 cells, a large amount of fluorescence was detected in the peri-nuclear cytoplasm. In rat neonatal myocardial cells, the sensitizer selectively localized in the large mitochondria. In both cell types, there was little phototoxicity when the peripheral cytoplasmic region was exposed, as compared to considerable phototoxicity with exposure of either the perinuclear or nuclear regions. CONCLUSION: Both the CPAE and PTK2 cells demonstrated that the nucleus followed by the perinuclear cytoplasm are the most sensitive cell areas with no sensitivity in the peripheral cytoplasm.

Mos oncogene product associates with kinetochores in mammalian somatic cells and disrupts mitotic progression.

The mos protooncogene has opposing effects on cell cycle progression. It is required for reinitiation of meiotic maturation and for meiotic progression through metaphase II, yet it is an active component of cytostatic factor. mos is a potent oncogene in fibroblasts, but high levels of expression are lethal. The lethality of mos gene expression in mammalian cells could be a consequence of a blockage induced by its cytostatic factor-related activity, which may appear at high dosage in mitotic cells. We have directly tested whether expression of the Mos protein can block mitosis in mammalian cells by microinjecting a fusion protein between Escherichia coli maltose-binding protein and Xenopus c-Mos into PtK1 epithelial cells and analyzing the cells by video time-lapse and immunofluorescence microscopy. Time-course analyses showed that Mos blocked mitosis by preventing progression to a normal metaphase. Chromosomes frequently failed to attain a bipolar orientation and were found near one pole. Injection of a kinase-deficient mutant Mos had no effect on mitosis, indicating that the blockage of mitotic progression required Mos kinase activity. Antitubulin immunostaining of cells blocked by Mos showed that microtubules were present but that spindle morphology was abnormal. Immunostaining for the Mos fusion protein showed that both wild-type and kinase mutant proteins localized at the kinetochores. Our results suggest that mitotic blockage by Mos may result from an action of the Mos kinase on the kinetochores, thus increasing chromosome instability and preventing normal congression.