Ron Balczon

The centrosome in animal cells and its functional homologs in plant and yeast cells.

The centrosome is the principal microtubule-organizing center in mammalian cells. Until recently, the centrosome could only be studied at the ultrastructural level and defined as a functional entity. However, during the past decade a number of clever experimental strategies have been used to identify numerous molecular components of the centrosome. The identification of biochemical subunits of the centrosome complex has allowed the centrosome to be investigated in much more detail, resulting in important advances being made in our understanding of microtubule nucleation events, spindle formation, the assembly and replication of the centrosome, and the nature of the microtubule-organizing centers in plant cells and lower eukaryotes. The next several years should see additional rapid progress in our understanding of the microtubule cytoskeleton as investigators begin to assign functions to the centrosome proteins that have already been reported and as additional centrosome components are discovered.

Role for microtubules in centrosome doubling in chinese hamster ovary cells

The centrosome must be replicated once, and only once, during each cell cycle. To achieve this somatic cells need to synthesize centrosome proteins, target those centrosome proteins to the parental centrosome, and then assemble the centrosome subunits into a functional organelle. The mechanisms that underlie each of these processes are not known. Studies were performed to investigate whether cellular microtubules are involved in centrosome doubling events. For these experiments, CHO cells were arrested in either hydroxyurea (HU) alone or in HU plus a microtubule inhibitor for 3640 h. The cells then were induced to enter mitosis and the numbers of spindle poles/centrosomes were counted following processing of the cells for immunofluorescence microscopy using anticentrosome antiserum. These studies demonstrated that centrosome replication events occurred in cells arrested with either HU alone or HU and taxol while centrosome replication did not occur in cells treated with HU and either nocodazole or colcemid. Immunoblot analysis determined that centrosome proteins were synthesized in HU/nocodazole-arrested cells and demonstrated that the role of microtubules in the centrosome replication process is not to ensure the synthesis of centrosome subunits. Rather, our results suggest that microtubules may be involved in the transport/targeting of centrosome subunits to the parental centrosome during duplication events. For microtubules to contribute to the transport of centrosome subunits during centrosome doubling, centrosome subunits would need to be able to bind to microtubules. To test this, co-sedimentation studies were performed and it was determined that the centrosome proteins, though overproduced under these conditions, remained soluble in HU/nocodazole-treated cells and co-pelleted with taxol-stabilized microtubules in the presence of GTP and AMP-PNP. Moreover, co-sedimentation of one of the centrosome proteins, PCM-1, with microtubules could be inhibited by pre-incubation of extracts with antibodies against dynactin. Together, these data suggest that during centrosome replication in somatic mammalian cells, PCM-1, and perhaps other centrosome components, are targeted to the centrosome via transport along microtubules by motor complexes that include dynein/dynactin.

Human Pulmonary Microvascular Endothelial Cells Support Productive Replication of Highly Pathogenic Avian Influenza Viruses: Possible Involvement in the Pathogenesis of Human H5N1 Virus Infection

ABSTRACT Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to cause sporadic human infections with a high fatality rate. Respiratory failure due to acute respiratory distress syndrome (ARDS) is a complication among hospitalized patients. Since progressive pulmonary endothelial damage is the hallmark of ARDS, we investigated host responses following HPAI virus infection of human pulmonary microvascular endothelial cells. Evaluation of these cells for the presence of receptors preferred by influenza virus demonstrated that avian-like (α2-3-linked) receptors were more abundant than human-like (α2-6-linked) receptors. To test the permissiveness of pulmonary endothelial cells to virus infection, we compared the replication of selected seasonal, pandemic (2009 H1N1 and 1918), and potentially pandemic (H5N1) influenza virus strains. We observed that these cells support productive replication only of HPAI H5N1 viruses, which preferentially enter through and are released from the apical surface of polarized human endothelial monolayers. Furthermore, A/Thailand/16/2004 and A/Vietnam/1203/2004 (VN/1203) H5N1 viruses, which exhibit heightened virulence in mammalian models, replicated to higher titers than less virulent H5N1 strains. VN/1203 infection caused a significant decrease in endothelial cell proliferation compared to other subtype viruses. VN/1203 virus was also found to be a potent inducer of cytokines and adhesion molecules known to regulate inflammation during acute lung injury. Deletion of the H5 hemagglutinin (HA) multibasic cleavage site did not affect virus infectivity but resulted in decreased virus replication in endothelial cells. Our results highlight remarkable tropism and infectivity of the H5N1 viruses for human pulmonary endothelial cells, resulting in the potent induction of host inflammatory responses.

Suppression of the Expression of a Pancreatic β-Cell Form of the Kinesin Heavy Chain by Antisense Oligonucleotides Inhibits Insulin Secretion from Primary Cultures of Mouse β-Cells1

Granular/vesicular transport is thought to be supported by microtubule-based force-generating adenosine triphosphatases such as kinesin. Kinesin is a motor molecule that has been well studied in brain and other neuronal tissues. Although vesicular transport is important for pancreatic beta-cell secretory activities, the role of kinesin in beta-cell function has not been investigated. It is hypothesized that kinesin functions as a translocator that associates with both microtubules and insulin-containing granules in beta-cells and transports the secretory granules from deep within the cytoplasm, where insulin is synthesized and processed, to the surface of beta-cells upon secretory stimulation. To test this hypothesis, a mouse beta-cell kinesin heavy chain complementary DNA was cloned and sequenced. Kinesin expression in primary cultures of mouse beta-cells then was selectively suppressed by antimouse beta-cell kinesin heavy chain antisense oligonucleotide treatment. Analysis of insulin secretion determined that the basal level of insulin secretion from the treated cells was decreased by 50%. Furthermore, glucose-stimulated insulin release from treated beta-cells was reduced by almost 70% after suppression of kinesin expression by antisense treatment. The findings from this study provide the first direct evidence that kinesin, a microtubule-based motor protein, plays an important role in insulin secretion.

Pseudomonas aeruginosa exotoxin Y is a promiscuous cyclase that increases endothelial Tau phosphorylation and permeability

Background: ExoY induces inter-endothelial gaps, although the mechanisms by which this occurs are poorly understood. Results: ExoY synthesized cAMP and cGMP, which caused endothelial Tau hyperphosphorylation, accumulation of insoluble Tau, inter-endothelial cell gaps, and increased permeability. Conclusion: ExoY is a promiscuous cyclase and an edema factor. Significance: Acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases. Exotoxin Y (ExoY) is a type III secretion system effector found in ∼ 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY causes inter-endothelial gaps and vascular leak, the mechanisms by which this occurs are poorly understood. Using both a bacteria-delivered and a codon-optimized conditionally expressed ExoY, we report that this toxin is a dual soluble adenylyl and guanylyl cyclase that results in intracellular cAMP and cGMP accumulation. The enzymatic activity of ExoY caused phosphorylation of endothelial Tau serine 214, accumulation of insoluble Tau, inter-endothelial cell gap formation, and increased macromolecular permeability. To discern whether the cAMP or cGMP signal was responsible for Tau phosphorylation and barrier disruption, pulmonary microvascular endothelial cells were engineered for the conditional expression of either wild-type guanylyl cyclase, which synthesizes cGMP, or a mutated guanylyl cyclase, which synthesizes cAMP. Sodium nitroprusside stimulation of the cGMP-generating cyclase resulted in transient Tau serine 214 phosphorylation and gap formation, whereas stimulation of the cAMP-generating cyclase induced a robust increase in Tau serine 214 phosphorylation, gap formation, and macromolecular permeability. These results indicate that the cAMP signal is the dominant stimulus for Tau phosphorylation. Hence, ExoY is a promiscuous cyclase and edema factor that uses cAMP and, to some extent, cGMP to induce the hyperphosphorylation and insolubility of endothelial Tau. Because hyperphosphorylated and insoluble Tau are hallmarks in neurodegenerative tauopathies such as Alzheimer disease, acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases.

The Pseudomonas aeruginosa exoenzyme Y impairs endothelial cell proliferation and vascular repair following lung injury

Exoenzyme Y (ExoY) is a Pseudomonas aeruginosa toxin that is introduced into host cells through the type 3 secretion system (T3SS). Once inside the host cell cytoplasm, ExoY generates cyclic nucleotides that cause tau phosphorylation and microtubule breakdown. Microtubule breakdown causes interendothelial cell gap formation and tissue edema. Although ExoY transiently induces interendothelial cell gap formation, it remains unclear whether ExoY prevents repair of the endothelial cell barrier. Here, we test the hypothesis that ExoY intoxication impairs recovery of the endothelial cell barrier following gap formation, decreasing migration, proliferation, and lung repair. Pulmonary microvascular endothelial cells (PMVECs) were infected with P. aeruginosa strains for 6 h, including one possessing an active ExoY (PA103 exoUexoT::Tc pUCPexoY; ExoY(+)), one with an inactive ExoY (PA103ΔexoUexoT::Tc pUCPexoY(K81M); ExoY(K81M)), and one that lacks PcrV required for a functional T3SS (ΔPcrV). ExoY(+) induced interendothelial cell gaps, whereas ExoY(K81M) and ΔPcrV did not promote gap formation. Following gap formation, bacteria were removed and endothelial cell repair was examined. PMVECs were unable to repair gaps even 3-5 days after infection. Serum-stimulated growth was greatly diminished following ExoY intoxication. Intratracheal inoculation of ExoY(+) and ExoY(K81M) caused severe pneumonia and acute lung injury. However, whereas the pulmonary endothelial cell barrier was functionally improved 1 wk following ExoY(K81M) infection, pulmonary endothelium was unable to restrict the hyperpermeability response to elevated hydrostatic pressure following ExoY(+) infection. In conclusion, ExoY is an edema factor that chronically impairs endothelial cell barrier integrity following lung injury.

Overexpression of cyclin A in human HeLa cells induces detachment of kinetochores and spindle pole/centrosome overproduction.

Abstract The combination of hydroxyurea (HU) and caffeine has been used for inducing kinetochore dissociation from mitotic chromosomes and for causing centrosome/spindle pole amplification. However, these effects on microtubule organizing centers (MTOCs) are limited to certain cell types. It was reasoned that if the biochemical differences in MTOC behavior between cells following HU treatment could be identified, then critical information concerning the regulation of these organelles would be obtained. During these studies, it was determined that cells from hamster, rat, and deer could be induced to enter mitosis with dissociated kinetochores and to synthesize centrosomes during arrest with HU, while cells from human and mouse could not. Comparisons between human HeLa cells and CHO cells determined that cyclin A levels were depressed in HeLa cells relative to CHO cells following HU addition. Overexpression of cyclin A in HeLa cells converted them to a cell type capable of detaching kinetochores from mitotic chromosomes. Ultrastructural analyses determined that the detached human kinetochores exhibited a normal plate-like morphology and appeared capable of associating with microtubules. In addition, HeLa cells overexpressing cyclin A also overproduced spindle poles during HU arrest, demonstrating that cyclin A activity also is important for centrosome replication during interphase. In summary, elevated cyclin A levels are important for the capacity of cells to be driven into mitosis by caffeine addition, for the ability of cells to progress to mitosis with detached kinetochores, and for centrosome/spindle pole replication.

Filamin A is a phosphorylation target of membrane but not cytosolic adenylyl cyclase activity

Transmembrane adenylyl cyclase (AC) generates a cAMP pool within the subplasma membrane compartment that strengthens the endothelial cell barrier. This cAMP signal is steered toward effectors that promote junctional integrity and is inactivated before it accesses microtubules, where the cAMP signal causes phosphorylation of tau, leading to microtubule disassembly and barrier disruption. During infection, Pseudomonas aeruginosa uses a type III secretion system to inject a soluble AC, ExoY, into the cytosol of pulmonary microvascular endothelial cells. ExoY generates a cAMP signal that disrupts the endothelial cell barrier. We tested the hypothesis that this ExoY-dependent cAMP signal causes phosphorylation of tau, without inducing phosphorylation of membrane effectors that strengthen endothelial barrier function. To approach this hypothesis, we first discerned the membrane compartment in which endogenous transmembrane AC6 resides. AC6 was resolved in caveolin-rich lipid raft fractions with calcium channel proteins and the cell adhesion molecules N-cadherin, E-cadherin, and activated leukocyte adhesion molecule. VE-cadherin was excluded from the caveolin-rich fractions and was detected in the bulk plasma membrane fractions. The actin binding protein, filamin A, was detected in all membrane fractions. Isoproterenol activation of ACs promoted filamin phosphorylation, whereas thrombin inhibition of AC6 reduced filamin phosphorylation within the membrane fraction. In contrast, ExoY produced a cAMP signal that did not cause filamin phosphorylation yet induced tau phosphorylation. Hence, our data indicate that cAMP signals are strictly compartmentalized; whereas cAMP emanating from transmembrane ACs activates barrier-enhancing targets, such as filamin, cAMP emanating from soluble ACs activates barrier-disrupting targets, such as tau.

Soluble adenylyl cyclase-dependent microtubule disassembly reveals a novel mechanism of endothelial cell retraction

Soluble adenylyl cyclase toxins, such as Pseudomonas aeruginosa exoY, generate a cAMP pool that retracts cell borders. However, the cytoskeletal basis by which this cAMP signal retracts cell borders is not known. We sought to determine whether activation of chimeric, soluble adenylyl cyclase I/II (sACI/II) reorganizes either microtubules or peripheral actin. Endothelial cells were stably transfected with either green fluorescent protein-labeled alpha-tubulin or beta-actin, and then infected with adenovirus to express sACI/II. Forskolin, which stimulates both the endogenously expressed transmembrane adenylyl cyclases and sACI/II, induced cell retraction accompanied by the reorganization of peripheral microtubules. However, cortical filamentous-actin (f-actin) did not reorganize into stress fibers, and myosin light-chain-20 phosphorylation was decreased. Isoproterenol, which activates endogenous adenylyl cyclases but does not activate sACI/II, did not induce endothelial cell gaps and did not influence microtubule or f-actin architecture. Thus, sACI/II generates a cAMP signal that reorganizes microtubules and induces cell retraction, without inducing f-actin stress fibers. These findings illustrate that endothelial cell gap formation can proceed without f-actin stress fiber formation, and provide mechanistic insight how bacterial adenylyl cyclase toxins reorganize the cytoskeleton to induce cell rounding.

Cold exposure reveals two populations of microtubules in pulmonary endothelia

Microtubules are composed of α-tubulin and β-tubulin dimers. Microtubules yield tubulin dimers when exposed to cold, which reassemble spontaneously to form microtubule fibers at 37°C. However, mammalian neurons, glial cells, and fibroblasts have cold-stable microtubules. While studying the microtubule toxicity mechanisms of the exotoxin Y from Pseudomonas aeruginosa in pulmonary microvascular endothelial cells, we observed that some endothelial microtubules were very difficult to disassemble in the cold. As a consequence, we designed studies to test the hypothesis that microvascular endothelium has a population of cold-stable microtubules. Pulmonary microvascular endothelial cells and HeLa cells (control) were grown under regular cell culture conditions, followed by exposure to an ice-cold water bath and a microtubule extraction protocol. Polymerized microtubules were detected by immunofluorescence confocal microscopy and Western blot analyses. After cold exposure, immunofluorescence revealed that the majority of HeLa cell microtubules disassembled, whereas a smaller population of endothelial cell microtubules disassembled. Immunoblot analyses showed that microvascular endothelial cells express the microtubule cold-stabilizing protein N-STOP (neuronal stable tubule-only polypeptides), and that N-STOP binds to endothelial microtubules after cold exposure, but not if microtubules are disassembled with nocodazole before cold exposure. Hence, pulmonary endothelia have a population of cold-stable microtubules.