A Novel Form of Rotavirus NSP2 and Phosphorylation-Dependent NSP2-NSP5 Interactions Are Associated with Viroplasm Assembly

Rotavirus (RV) replication occurs in cytoplasmic inclusions called viroplasms whose formation requires the interactions of RV proteins NSP2 and NSP5; however, the specific role(s) of NSP2 in viroplasm assembly remains largely unknown. To study viroplasm formation in the context of infection, we characterized two new monoclonal antibodies (MAbs) specific for NSP2. These MAbs show high-affinity binding to NSP2 and differentially recognize distinct pools of NSP2 in RV-infected cells; a previously unrecognized cytoplasmically dispersed NSP2 (dNSP2) is detected by an N-terminal binding MAb, and previously known viroplasmic NSP2 (vNSP2) is detected by a C-terminal binding MAb. Kinetic experiments in RV-infected cells demonstrate that dNSP2 is associated with NSP5 in nascent viroplasms that lack vNSP2. As viroplasms mature, dNSP2 remains in viroplasms, and the amount of diffuse cytoplasmic dNSP2 increases. vNSP2 is detected in increasing amounts later in infection in the maturing viroplasm, suggesting a conversion of dNSP2 into vNSP2. Immunoprecipitation experiments and reciprocal Western blot analysis confirm that there are two different forms of NSP2 that assemble in complexes with NSP5, VP1, VP2, and tubulin. dNSP2 associates with hypophosphorylated NSP5 and acetylated tubulin, which is correlated with stabilized microtubules, while vNSP2 associates with hyperphosphorylated NSP5. Mass spectroscopy analysis of NSP2 complexes immunoprecipitated from RV-infected cell lysates show both forms of NSP2 are phosphorylated, with a greater proportion of vNSP2 being phosphorylated compared to dNSP2. Together, these data suggest that dNSP2 interacts with viral proteins, including hypophosphorylated NSP5, to initiate viroplasm formation, while viroplasm maturation includes phosphorylation of NSP5 and vNSP2.     

Household Catastrophic Healthcare Expenditure and Impoverishment Due to Rotavirus Gastroenteritis Requiring Hospitalization in Malaysia

Background

While healthcare costs for rotavirus gastroenteritis requiring hospitalization may be burdensome on households in Malaysia, exploration on the distribution and catastrophic impact of these expenses on households are lacking.

Objectives

We assessed the economic burden, levels and distribution of catastrophic healthcare expenditure, the poverty impact on households and inequities related to healthcare payments for acute gastroenteritis requiring hospitalization in Malaysia.

Methods

A two-year prospective, hospital-based study was conducted from 2008 to 2010 in an urban (Kuala Lumpur) and rural (Kuala Terengganu) setting in Malaysia. All children under the age of 5 years admitted for acute gastroenteritis were included. Patients were screened for rotavirus and information on healthcare expenditure was obtained.

Results

Of the 658 stool samples collected at both centers, 248 (38%) were positive for rotavirus. Direct and indirect costs incurred were significantly higher in Kuala Lumpur compared with Kuala Terengganu (US$222 Vs. US$45; p<0.001). The mean direct and indirect costs for rotavirus gastroenteritis consisted 20% of monthly household income in Kuala Lumpur, as compared with only 5% in Kuala Terengganu. Direct medical costs paid out-of-pocket caused 141 (33%) households in Kuala Lumpur to experience catastrophic expenditure and 11 (3%) households to incur poverty. However in Kuala Terengganu, only one household (0.5%) experienced catastrophic healthcare expenditure and none were impoverished. The lowest income quintile in Kuala Lumpur was more likely to experience catastrophic payments compared to the highest quintile (87% vs 8%). The concentration index for out-of-pocket healthcare payments was closer to zero at Kuala Lumpur (0.03) than at Kuala Terengganu (0.24).

Conclusions

While urban households were wealthier, healthcare expenditure due to gastroenteritis had more catastrophic and poverty impact on the urban poor. Universal rotavirus vaccination would reduce both disease burden and health inequities in Malaysia.     

Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails

Microtubule dynamics in cells are regulated by associated proteins that can be either stabilizers or destabilizers. A class of destabilizers that is important in a large number of cellular activities is the microtubule-severing enzymes, yet little is known about how they function. Katanin p60 was the first ATPase associated with microtubule severing. Here, we investigate the activity of katanin severing using a GFP-labeled human version. We quantify the effect of katanin concentration on katanin binding and severing activity. We find that free tubulin can inhibit severing activity by interfering with katanin binding to microtubules. The inhibition is mediated by the sequence of the tubulin and specifically depends on the carboxy-terminal tails. We directly investigate the inhibition effect of tubulin carboxy-terminal tails using peptide sequences of α-, β-, or detyrosinated α-tubulin tails that have been covalently linked to bovine serum albumin. Our results show that β-tubulin tails are the most effective at inhibiting severing, and that detyrosinated α-tubulin tails are the least effective. These results are distinct from those for other severing enzymes and suggest a scheme for regulation of katanin activity in cells dependent on free tubulin concentration and the modification state of the tubulin.     

Type D Retrovirus Capsid Assembly and Release Are Active Events Requiring ATP

Mason-Pfizer monkey virus (M-PMV), the prototype type D retrovirus, differs from most other retroviruses by assembling its Gag polyproteins into procapsids in the cytoplasm of infected cells. Once assembled, the procapsids migrate to the plasma membrane, where they acquire their envelope during budding. Because the processes of M-PMV protein transport, procapsid assembly, and budding are temporally and spatially unlinked, we have been able to determine whether cellular proteins play an active role during the different stages of procapsid morphogenesis. We report here that at least two stages of morphogenesis require ATP. Both procapsid assembly and procapsid transport to the plasma membrane were reversibly blocked by treating infected cells with sodium azide and 2-deoxy-d-glucose, which we show rapidly and reversibly depletes cellular ATP pools. Assembly of procapsids in vitro in a cell-free translation/assembly system was inhibited by the addition of nonhydrolyzable ATP analogs, suggesting that ATP hydrolysis and not just ATP binding is required. Since retrovirus Gag polyproteins do not bind or hydrolyze ATP, these results demonstrate that cellular components must play an active role during retrovirus morphogenesis.

Assembly and release of nascent retrovirus particles requires that the viral precursor polyproteins and genomic RNAs, and certain host cell tRNAs, migrate to the plasma membrane, where budding occurs. Two discrete intracellular transport pathways are utilized during the assembly of the infectious virion. The viral glycoproteins are synthesized on membrane-bound polysomes and are transported through the secretory pathway of the cell to the plasma membrane, where they colocalize with the immature capsid during the budding process (20). The major structural proteins of the viral capsid and the enzymatic proteins are synthesized in the cytoplasm on free polysomes and are transported to the underside of the plasma membrane (13, 36). While many of the details of the secretory pathway have been established, the mechanisms for intracytoplasmic protein transport are poorly understood.The major structural polyprotein (Gag) of a nascent retrovirus capsid is encoded by the gag gene. Unlike most enveloped RNA viruses in which the viral glycoproteins mediate assembly by stabilizing the interactions between the capsid proteins and the viral membrane, retroviral Gag proteins can drive capsid assembly and budding in the absence of all the other viral gene products (19, 55, 58). As such, they contain all cis-acting information necessary for intracytoplasmic transport, capsid assembly, membrane binding, envelopment, and release from the cell surface. Assembly of the immature retrovirus capsid begins shortly after the Gag polyproteins are synthesized and modified by myristylation (15, 17, 40, 47–49). The Gag proteins of most retroviruses (the type C avian and mammalian viruses, lentiviruses, and human T-cell leukemia virus/bovine leukemia virus-related viruses) migrate directly to the plasma membrane, where they coalesce into spherical, immature capsids and simultaneously bud through the lipid bilayer, thereby acquiring their envelope. During or shortly after release, the Gag protein is cleaved by the viral protease into the internal structural (NH₂-MA [matrix], CA [capsid], and NC [nucleocapsid]) proteins of the mature, infectious virion (22). In contrast, the Gag proteins of the mammalian and type B and D viruses (mouse mammary tumor virus [MMTV] and Mason-Pfizer monkey virus [M-PMV], respectively) accumulate in the cytoplasm, where they assemble into spherical structures in the absence of membranes. These nascent particles have been referred to as intracytoplasmic type A particles, but by analogy to other viruses and bacteriophages, we have redefined them as procapsids (55). Once assembled, procapsids are transported to the plasma membrane, from which they bud. Despite the different assembly strategies, the processes whereby Gag proteins assemble into procapsids are probably similar since a single amino acid change near the amino terminus of the Gag protein from M-PMV has been shown to convert it to the type C morphogenic pathway (41).Genetic analyses of the gag genes from different retroviruses have shown that Gag proteins contain specific domains which are required for capsid formation. A membrane binding (M) domain has been located at the amino-terminal end of Gag of several retroviruses (31, 43, 60, 61). A late (L) domain functions during the budding and release. In Rous sarcoma virus (RSV) and M-PMV, the L domain is located between the MA and CA domains (57, 59). An equivalent domain in the lentiviruses has been found near the carboxy terminus of the Gag precursor (34). A third domain (I), located near the CA-NC junction, appears to be a region of interaction between Gag proteins (3, 56). Despite the lack of any extensive sequence similarities between different Gag proteins, there is functional conservation between assembly domains. Chimeric Gag proteins containing the M, L, and I domains from different retroviruses can assemble into capsid-like structures and mediate budding at the plasma membrane (3, 9, 10, 34).The M-PMV Gag protein contains additional assembly elements which influence procapsid assembly, stability, and transport. This virus contains a region within Gag (known as p12) that is not found in either the type C viruses or lentiviruses. It has been suggested from biochemical data derived from studies with p12 deletion mutants that this domain assists in assembly by stabilizing intermolecular Gag associations (50). Protein stability and protein/procapsid transport depend on sequences in the MA domain which appear to be distinct from the M domain. As mentioned above, a single point mutation in MA at residue 55 results in a Gag protein that no longer assembles in the cytoplasm but rather assembles at the plasma membrane. This mutation lies within an 18-amino-acid region of the MA domain that has sequence similarity only to the type B retroviruses (41). The nuclear magnetic resonance-derived solution structure of a nonmyristylated M-PMV MA protein indicates that this region folds into a structured turn which is solvent accessible in the monomer and trimer models (8). Moreover, this structural feature is absent in human immunodeficiency virus (HIV), simian immunodeficiency virus, human T-cell leukemia virus, and bovine leukemia virus MA proteins (7, 18, 27–30, 37). It is reasonable, therefore, to suspect that this region contains a cytoplasmic protein transport signal which must interact with a cellular factor. In contrast, other mutations in either the myristic acid addition signal or at a variety of positions elsewhere in the MA coding region result in Gag proteins that fail to be released as virus-like particles despite assembling into procapsids in the cytoplasm (40, 43). Thus, the M-PMV Gag protein appears to contain a second cytoplasmic transport signal which normally directs assembled procapsids and not unassembled Gag proteins to the plasma membrane. It is implied in this model that the M-PMV Gag protein must utilize multiple cellular components during the different stages of assembly and release.The type D retroviruses provide a useful system for studying morphogenic events since procapsid assembly, protein transport, and budding are temporally and spatially unlinked. We report here that in infected cells and an in vitro translation/assembly system, procapsid assembly and transport to the plasma membrane require ATP. Thus, cellular proteins do play an active role during at least two stages of M-PMV morphogenesis.     

Microtubules Regulate Localization and Availability of Insulin Granules in Pancreatic Beta Cells

Two key prerequisites for glucose-stimulated insulin secretion (GSIS) in β cells are the proximity of insulin granules to the plasma membrane and their anchoring or docking to the plasma membrane (PM). Although recent evidence has indicated that both of these factors are altered in the context of diabetes, it is unclear what regulates localization of insulin granules and their interactions with the PM within single cells. Here, we demonstrate that microtubule (MT)-motor-mediated transport dynamics have a critical role in regulating both factors. Super-resolution imaging shows that whereas the MT cytoskeleton resembles a random meshwork in the cells’ interior, MTs near the cell surface are preferentially aligned with the PM. Computational modeling suggests two consequences of this alignment. First, this structured MT network preferentially withdraws granules from the PM. Second, the binding and transport of insulin granules by MT motors prevents their stable anchoring to the PM. These findings suggest the MT cytoskeleton may negatively regulate GSIS by both limiting the amount of insulin proximal to the PM and preventing or breaking interactions between the PM and the remaining nearby insulin granules. These results predict that altering MT network structure in β cells can be used to tune GSIS. Thus, our study points to the potential of an alternative therapeutic strategy for diabetes by targeting specific MT regulators.     

Lotus japonicus CASTOR and POLLUX Are Ion Channels Essential for Perinuclear Calcium Spiking in Legume Root Endosymbiosis

The mechanism underlying perinuclear calcium spiking induced during legume root endosymbioses is largely unknown. Lotus japonicus symbiosis-defective castor and pollux mutants are impaired in perinuclear calcium spiking. Homology modeling suggested that the related proteins CASTOR and POLLUX might be ion channels. Here, we show that CASTOR and POLLUX form two independent homocomplexes in planta. CASTOR reconstituted in planar lipid bilayers exhibited ion channel activity, and the channel characteristics were altered in a symbiosis-defective mutant carrying an amino acid replacement close to the selectivity filter. Permeability ratio determination and competition experiments reveled a weak preference of CASTOR for cations such as potassium over anions. POLLUX has an identical selectivity filter region and complemented a potassium transport–deficient yeast mutant, suggesting that POLLUX is also a potassium-permeable channel. Immunogold labeling localized the endogenous CASTOR protein to the nuclear envelope of Lotus root cells. Our data are consistent with a role of CASTOR and POLLUX in modulating the nuclear envelope membrane potential. They could either trigger the opening of calcium release channels or compensate the charge release during the calcium efflux as counter ion channels.     

Myosin-Va and Dynamic Actin Oppose Microtubules to Drive Long-Range Organelle Transport

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The Orientation and Localization of Cortical Microtubules in Differentiating Conifer Tracheids during Cell Expansion

Arrays of cortical microtubules (MTs) on radial walls in differentiatingtracheids of Taxus cuspidata were randomly oriented when primarywalls formed. The orientation of MTs changed progressively fromlongitudinal to transverse as cells expanded. During formationof primary walls, MTs in differentiating tracheids disappearedlocally at sites of future intertracheal bordered pits. In furtherdifferentiated tracheids, circular bands of MTs were observedaround the edges of developing bordered pits. (Received July 17, 1996; Accepted November 11, 1996)     

Fusion between Perinuclear Virions and the Outer Nuclear Membrane Requires the Fusogenic Activity of Herpes Simplex Virus gB

Herpesviruses cross nuclear membranes (NMs) in two steps, as follows: (i) capsids assemble and bud through the inner NM into the perinuclear space, producing enveloped virus particles, and (ii) the envelopes of these virus particles fuse with the outer NM. Two herpes simplex virus (HSV) glycoproteins, gB and gH (the latter, likely complexed as a heterodimer with gL), are necessary for the second step of this process. Mutants lacking both gB and gH accumulate in the perinuclear space or in herniations (membrane vesicles derived from the inner NM). Both gB and gH/gL are also known to act directly in fusing the virion envelope with host cell membranes during HSV entry into cells, i.e., both glycoproteins appear to function directly in different aspects of the membrane fusion process. We hypothesized that HSV gB and gH/gL also act directly in the membrane fusion that occurs during virus egress from the nucleus. Previous studies of the role of gB and gH/gL in nuclear egress involved HSV gB and gH null mutants that could potentially also possess gross defects in the virion envelope. Here, we produced recombinant HSV-expressing mutant forms of gB with single amino acid substitutions in the hydrophobic “fusion loops.” These fusion loops are thought to play a direct role in membrane fusion by insertion into cellular membranes. HSV recombinants expressing gB with any one of four fusion loop mutations (W174R, W174Y, Y179K, and A261D) were unable to enter cells. Moreover, two of the mutants, W174Y and Y179K, displayed reduced abilities to mediate HSV cell-to-cell spread, and W174R and A261D exhibited no spread. All mutant viruses exhibited defects in nuclear egress, enveloped virions accumulated in herniations and in the perinuclear space, and fewer enveloped virions were detected on cell surfaces. These results support the hypothesis that gB functions directly to mediate the fusion between perinuclear virus particles and the outer NM.Herpesvirus glycoproteins gB and gH/gL participate in two separate membrane fusion events that occur during different stages of virus replication. First, during virus entry into cells, gB and gH/gL promote fusion between the virion envelope and either the plasma membrane or endosomes (reviewed in references 6, 21, 27, and 39). Second, herpes simplex virus (HSV) gB and gH (likely complexed to form a heterodimer with gL), and likely homologues in other herpesviruses, promote nuclear egress (12). Herpesvirus capsids are produced in the nucleus and cross the nuclear envelope (NE) by envelopment at the inner nuclear membrane (NM), producing perinuclear virions that then fuse with the outer NM (reviewed in references 35 and 36). There is evidence that HSV gB and gH/gL function in a redundant fashion in fusion between enveloped, perinuclear virus particles and the outer NM (12), whereas both gB and gH/gL are essential for entry fusion (8, 13, 38). Much more is known about the mechanisms involved in entry fusion than those involved in egress fusion, and many important questions remain in terms of how these two membrane fusion processes relate to each other.Entry of HSV into cells involves interactions between the viral receptor-binding protein gD and the gD receptors (16, 28, 30, 37). When gD binds to its receptors, there are conformational changes in gD which apparently activate gB and gH/gL, so that these glycoproteins promote fusion involving the virion envelope and cellular membranes (21, 32). By using split green fluorescent protein fusion proteins, also denoted bimolecular complementation, two groups showed that gD binding to gD ligands triggers interactions between gB and gH/gL and that this is accompanied by cell-cell fusion (1, 2). There is also evidence that gB and gH/gL contribute to different stages of membrane fusion. When gH/gL is expressed with gD, there is hemifusion (mixing of the outer leaflets of membranes) of adjacent cells, and this partial fusion is apparently mediated by gH/gL (41). However, full fusion (mixing of both inner and outer leaflets) occurs only when gB is coexpressed with gD and gH/gL (41). Also supporting a role for gH in membrane fusion, peptides based on heptad repeats in gH can disrupt model membranes (14, 15, 17). HSV gB is a class III fusion protein, structurally similar to vesicular stomatitis virus G protein, with a three-stranded coil-coil barrel in the central region of the molecule reminiscent of class I fusion proteins, e.g., influenza virus hemagglutinin (22). Therefore, herpesvirus gB and gH/gL differ substantially from the fusion proteins expressed by all other well-studied viruses because both gB and gH/gL participate directly in membrane fusion, apparently functioning in different aspects of entry fusion.HSV gB and other viral class III fusion proteins differ from class I fusion proteins that have N-terminal, hydrophobic fusion peptides because class III fusion proteins possess internal bipartite “fusion loops” composed of both hydrophobic and hydrophilic residues (3, 22). In the solved structure of the HSV gB ectodomain, which might represent a postfusion form of the protein, the fusion loops are located near the base of the molecule, adjacent to the virion envelope (22). Mutant forms of gB with single amino acid substitutions in these fusion loops displayed diminished cell-cell fusion activity when transfected into cells with gD and gH/gL (20). Cell-cell fusion approximates the fusion that occurs during entry, defining the minimal fusion machinery, although there are differences between entry and cell-cell fusion (10). Moreover, full-length gB molecules with fusion loop mutations failed to complement gB null HSV (19). Recently, it was demonstrated that the HSV gB extracellular domain can interact with liposomes in vitro and that this binding depends upon gB''s fusion loops (19).Herpesvirus capsids are assembled in the nucleus and acquire an envelope by budding through the inner NM. For a short time, enveloped virus particles are found in the space between the inner and outer NMs (perinuclear space), but then the envelopes of these particles fuse with the outer NM, releasing capsids into the cytoplasm (reviewed in references 35 and 36). Cytoplasmic capsids acquire a second envelope by budding into the trans-Golgi network, and this secondary envelopment involves redundant or additive functions of gE/gI and gD, i.e., either of these glycoproteins will suffice (11). The second step of the nuclear egress pathway involving membrane fusion between the envelope of perinuclear particles and the outer NM requires HSV glycoproteins gB and gH/gL (12). HSV double mutants lacking both gB and gH accumulate enveloped virus particles in the perinuclear space and in herniations, i.e., membrane vesicles that bulge into the nucleoplasm and derive from the inner NM (12). These observations, coupled with the evidence that gB and gH/gL are fusion proteins, suggested that gB and gH/gL promote the fusion between virus particles and the outer NM. However, there is one important difference between nuclear egress fusion and entry fusion. Virus mutants lacking either gB or gH are unable to enter cells, but such mutants have fewer defects in nuclear egress than double mutants lacking both gB and gH (12). Thus, as with secondary envelopment that involves gD and gE/gI, glycoproteins gB and gH/gL act in a redundant or additive fashion to mediate the fusion between the envelope of perinuclear virus particles and the outer NM. It is also important to note that there appear to be other mechanisms by which HSV particles can exit the perinuclear space. For example, although a substantial number of gB gH null double mutants accumulated in herniations (increased by ∼10-fold), some virions were seen on cell surfaces, although their numbers were reduced by ∼2.5- to 5-fold compared with those of wild-type HSV (12, 46).HSV entry fusion is triggered by gD binding to one of its ligands. However, it is not clear what triggers fusion of the envelope of perinuclear particles with the outer NM. gD, gB, gH, gM, gK, and other viral membrane proteins are all present in NMs and in perinuclear virus particles (4, 12, 25, 40, 42, 44). It seems unlikely that there are substantial quantities of known gD receptors in NMs, although this has not been carefully examined and there may well be unidentified gD receptors present in NMs. However, if fusion at NMs is not activated by gD binding to gD receptors, there must be other mechanisms to trigger this fusion. There is evidence that HSV gK negatively regulates fusion at the NE because (i) overexpression of gK causes enveloped virus particles to accumulate in the perinuclear space (25) and (ii) gK is primarily localized to the endoplasmic reticulum and NM and is not substantially found in extracellular virions (26, 34). Another potential regulatory mechanism for fusion at the outer NM involves phosphorylation of the cytoplasmic domain of gB by the HSV kinase US3 (46). An HSV recombinant lacking gH and expressing a mutant gB with a substitution, T887A, affecting an amino acid in the gB cytoplasmic domain displayed reduced US3-dependent phosphorylation and accumulated enveloped virus particles in herniations (46). This mutation in gB did not alter HSV entry into cells (31, 46). Together, these results suggest that HSV fusion with the outer NM differs from entry fusion in some, but likely not all, important mechanistic details.Given that both gB and gH/gL are well established as fusion proteins for virus entry, we hypothesized that these glycoproteins directly mediate the membrane fusion that occurs between the envelope of perinuclear virus particles and the outer NM (12, 46). However, there are other possibilities. For example, it is conceivable that loss of both gB and gH alters the structure of the envelope of perinuclear HSV virions so that other HSV glycoproteins (that directly promote fusion) are affected. To address this issue and extend our understanding of how gB functions in nuclear egress fusion, we constructed HSV recombinants that express mutant forms of gB with substitutions in the fusion loops. These viruses also lacked gH, making nuclear egress totally dependent on a functional form of gB. By propagating these recombinants using gH-expressing cells, we could produce virus particles including gH and the mutant gB molecules. These HSV recombinants expressing gH as well as gB fusion loops, W174R, W174Y, Y179K, and A261D, were all unable to enter cells. However, two recombinants, expressing W174Y and Y179K, exhibited some cell-to-cell spread while the other two, expressing W174R and A261D, did not spread beyond single infected cells. All four recombinants infected into cells lacking gH exhibited defects in nuclear egress. These results provide strong support for the hypothesis that gB acts directly to mediate the fusion of the virion envelope with the outer NM during HSV egress.     

Mitochondrial Morphological Features Are Associated with Fission and Fusion Events

Mitochondria are dynamic organelles that undergo constant remodeling through the regulation of two opposing processes, mitochondrial fission and fusion. Although several key regulators and physiological stimuli have been identified to control mitochondrial fission and fusion, the role of mitochondrial morphology in the two processes remains to be determined. To address this knowledge gap, we investigated whether morphological features extracted from time-lapse live-cell images of mitochondria could be used to predict mitochondrial fate. That is, we asked if we could predict whether a mitochondrion is likely to participate in a fission or fusion event based on its current shape and local environment. Using live-cell microscopy, image analysis software, and supervised machine learning, we characterized mitochondrial dynamics with single-organelle resolution to identify features of mitochondria that are predictive of fission and fusion events. A random forest (RF) model was trained to correctly classify mitochondria poised for either fission or fusion based on a series of morphological and positional features for each organelle. Of the features we evaluated, mitochondrial perimeter positively correlated with mitochondria about to undergo a fission event. Similarly mitochondrial solidity (compact shape) positively correlated with mitochondria about to undergo a fusion event. Our results indicate that fission and fusion are positively correlated with mitochondrial morphological features; and therefore, mitochondrial fission and fusion may be influenced by the mechanical properties of mitochondrial membranes.     

Microtubules Are required for motility and positioning of vesicles and mitochondria in hyphal tip cells of Allomyces macrogynus

We have used video-enhanced light microscopy and digital image processing to characterize the intracellular motility and positioning of vesicles ( approximately 1-microm diameter) and mitochondria in growing hyphal tip cells of Allomyces macrogynus. These observations were coupled with cytoskeletal inhibitory experiments to define the roles of the microtubule and actin cytoskeletons in organelle translocation and positioning. Vesicles and mitochondria were abundant in apical and subapical hypha regions. Vesicles traveled along paths that were parallel to the longitudinal axis of the cell. Anterograde (i.e., toward the hyphal apex) and retrograde (i.e., away from the hyphal apex) movements of vesicles occurred at average rates of 4.0 and 2.2 microm/s, respectively. Bidirectional travel of vesicles along common paths was noted in the cortical cytoplasm. Mitochondria were aligned mostly parallel to the long axis of the hypha, except those extending into the hyphal apex, which were oriented toward the Spitzenk?rper. In regions of the subapical hypha mitochondria were often restricted to the cortical cytoplasm and nuclei occupied the central cytoplasmic region. Mitochondria displayed rapid anterograde movements reaching speeds of 3.0 microm/s, but primarily maintained a constant position relative to either the advancing cytoplasm or the lateral cell wall. Cytoskeletal disruption experiments showed that the positioning of mitochondria and motility of vesicles and mitochondria were microtubule-based and suggested that the actin cytoskeleton played uncertain roles.     

Tubulin Polyglycylation: Differential Posttranslational Modification of Dynamic Cytoplasmic and Stable Axonemal Microtubules in Paramecium

Polyglycylation, a posttranslational modification of tubulin, was discovered in the highly stable axonemal microtubules of Paramecium cilia where it involves the lateral linkage of up to 34 glycine units per tubulin subunit. The observation of this type of posttranslational modification mainly in axonemes raises the question as to its relationship with axonemal organization and with microtubule stability. This led us to investigate the glycylation status of cytoplasmic microtubules that correspond to the dynamic microtubules in Paramecium. Two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49, are shown here to exhibit different affinities toward mono- and polyglycylated synthetic tubulin peptides. Using immunoblotting and mass spectrometry, we show that cytoplasmic tubulin is glycylated. In contrast to the highly glycylated axonemal tubulin, which is recognized by the two mAbs, cytoplasmic tubulin reacts exclusively with TAP 952, and the α- and β- tubulin subunits are modified by only 1–5 and 2–9 glycine units, respectively. Our analyses suggest that most of the cytoplasmic tubulin contains side chain lengths of 1 or 2 glycine units distributed on several glycylation sites. The subcellular partition of distinct polyglycylated tubulin isoforms between cytoplasmic and axonemal compartments implies the existence of regulatory mechanisms for glycylation. By following axonemal tubulin immunoreactivity with anti-glycylated tubulin mAbs upon incubation with a Paramecium cellular extract, the presence of a deglycylation enzyme is revealed in the cytoplasm of this organism. These observations establish that polyglycylation is reversible and indicate that, in vivo, an equilibrium between glycylating and deglycylating enzymes might be responsible for the length of the oligoglycine side chains of tubulin.     

Convergence and Extrusion Are Required for Normal Fusion of the Mammalian Secondary Palate

The fusion of two distinct prominences into one continuous structure is common during development and typically requires integration of two epithelia and subsequent removal of that intervening epithelium. Using confocal live imaging, we directly observed the cellular processes underlying tissue fusion, using the secondary palatal shelves as a model. We find that convergence of a multi-layered epithelium into a single-layer epithelium is an essential early step, driven by cell intercalation, and is concurrent to orthogonal cell displacement and epithelial cell extrusion. Functional studies in mice indicate that this process requires an actomyosin contractility pathway involving Rho kinase (ROCK) and myosin light chain kinase (MLCK), culminating in the activation of non-muscle myosin IIA (NMIIA). Together, these data indicate that actomyosin contractility drives cell intercalation and cell extrusion during palate fusion and suggest a general mechanism for tissue fusion in development.     

Frequency and Selectivity of Mitochondrial Fusion Are Key to Its Quality Maintenance Function

Turnover of mitochondria by autophagy constitutes an essential quality maintenance mechanism. Recent studies have demonstrated that efficient clearance of damaged mitochondrial components depends on mitochondrial dynamics, a process characterized by frequent fusion and fission events that enable the redistribution of mitochondrial components across a population of hundreds of individual mitochondria. The presented simulation identifies kinetic parameters of fusion and fission that may influence the maintenance of mitochondrial function. The program simulated repetitive cycles of fusion and fission events in which intact and damaged mitochondrial contents were redistributed between fusion mates. Redistribution impacted mitochondrial function, thereby influencing the fate of each mitochondrion, to be either destined for a subsequent fusion or eliminated by autophagy. Our findings indicate that, when paired with fission, fusion events may serve to accelerate the removal of damaged mitochondrial components by autophagy. The model predicts the existence of an optimal frequency of fusion and fission events that can maintain respiratory function at steady-state levels amid the existence of a continuous damaging process that inactivates mitochondrial components. A further elevation of the fusion frequency can increase the clearance efficiency of damaged content. However, this requires fusion to be a selective process in which depolarized mitochondria are excluded from the fusing population. The selectivity of fusion was found to be particularly beneficial in conditions of elevated rate of damage, because it permits the increase of fusion frequency without compromising the removal of damaged content by autophagy.     

Visualization of the Peroxisomal Compartment in Living Mammalian Cells: Dynamic Behavior and Association with Microtubules

Peroxisomes in living CV1 cells were visualized by targeting the green fluorescent protein (GFP) to this subcellular compartment through the addition of a COOH-terminal peroxisomal targeting signal 1 (GFP–PTS1). The organelle dynamics were examined and analyzed using time-lapse confocal laser scanning microscopy. Two types of movement could be distinguished: a relatively slow, random, vibration-like movement displayed by the majority (~95%) of the peroxisomes, and a saltatory, fast directional movement displayed by a small subset (~5%) of the peroxisomes. In the latter instance, peak velocities up to 0.75 μm/s and sustained directional velocities up to 0.45 μm/s over 11.5 μm were recorded. Only the directional type of motion appeared to be energy dependent, whereas the vibrational movement continued even after the cells were depleted of energy. Treatment of cells, transiently expressing GFP–PTS1, with microtubule-destabilizing agents such as nocodazole, vinblastine, and demecolcine clearly altered peroxisome morphology and subcellular distribution and blocked the directional movement. In contrast, the microtubule-stabilizing compound paclitaxel, or the microfilament-destabilizing drugs cytochalasin B or D, did not exert these effects. High resolution confocal analysis of cells expressing GFP–PTS1 and stained with anti-tubulin antibodies revealed that many peroxisomes were associated with microtubules. The GFP–PTS1–labeled peroxisomes were found to distribute themselves in a stochastic, rather than ordered, manner to daughter cells at the time of mitosis.     

Viruses and Cells with Mutations Affecting Viral Entry Are Selected during Persistent Rotavirus Infections of MA104 Cells

To better understand mechanisms of persistent rotavirus infections of cultured cells, we established independent, persistently infected cultures of MA104 cells, using rotavirus strain SA11. The cultures were either passaged when the cells reached confluence or supplemented with fresh medium every 7 days. Viral titers in culture lysates varied from 10⁴ to 10⁷ PFU per ml during 350 days of culture maintenance. Trypan blue staining indicated that 72 to 100% of cells in the cultures were viable, and immunocytochemical staining using a monoclonal antibody directed against viral protein VP6 demonstrated that 38 to 63% of the cells contained rotavirus antigen. We tested the capacity of rotaviruses isolated from the persistently infected cultures (PI viruses) to infect cells cured of persistent infection. Although wild-type (wt) and PI viruses produced equivalent yields in parental MA104 cells, PI viruses produced greater yields than wt virus in cured cells, which indicates that viruses and cells coevolve during persistent rotavirus infections of MA104 cells. To determine whether mutations in viruses and cells selected during these persistent infections affect viral entry, we tested the effect of trypsin treatment of the viral inoculum on growth of wt and PI viruses. Trypsin pretreatment is required for postattachment penetration of rotavirus virions into cells. In contrast to the case with wt virus, PI viruses produced equivalent yields with and without trypsin pretreatment in parental MA104 cells. However, PI viruses required trypsin pretreatment for efficient growth in cured cells. These results indicate that mutant viruses and cells are selected during maintenance of persistent rotavirus infections of MA104 cells and suggest that mutations in each affect trypsin-dependent steps in rotavirus entry.     

轮状病毒肠炎患儿T细胞和红细胞免疫功能动态观察

报道４０例轮状病毒肠炎患儿急性期和恢复期外周血Ｔ细胞亚群和红细胞免疫功能的变化,并与同龄正常儿童进行了比较。结果表明急性期患儿ＣＤ３、ＣＤ４细胞及ＣＤ４／ＣＤ８比值均明显降低,ＣＤ８不变,至恢复期随着临床症状的改善,ＣＤ３、ＣＤ４及ＣＤ４／ＣＤ８逐渐正常。轮状病毒肠炎患儿急性期红细胞Ｃ３ｂ受体花环率显著下降,恢复期接近正常。结果提示细胞免疫参与了轮状病毒肠炎的感染过程,ＣＤ４／ＣＤ８与该病的预后有关。轮状病毒肠炎急性期可有继发性红细胞免疫功能下降,治疗时,有必要维持患儿的红细胞免疫功能,以提高其抗感染能力