Wolbachia-Host Interactions: Host Mating Patterns Affect Wolbachia Density Dynamics

Wolbachia are maternally inherited intracellular bacteria that infect a wide range of arthropods and cause an array of effects on host reproduction, fitness and mating behavior. Although our understanding of the Wolbachia-associated effects on hosts is rapidly expanding, our knowledge of the host factors that mediate Wolbachia dynamics is rudimentary. Here, we explore the interactions between Wolbachia and its host, the two-spotted spider mite Tetranychus urticae Koch. Our results indicate that Wolbachia induces strong cytoplasmic incompatibility (CI), increases host fecundity, but has no effects on the longevity of females and the mating competitiveness of males in T. urticae. Most importantly, host mating pattern was found to affect Wolbachia density dynamics during host aging. Mating of an uninfected mite of either sex with an infected mite attenuates the Wolbachia density in the infected mite. According to the results of Wolbachia localization, this finding may be associated with the tropism of Wolbachia for the reproductive tissue in adult spider mites. Our findings describe a new interaction between Wolbachia and their hosts.     

Reciprocal Intramolecular Interactions of Tomosyn Control Its Inhibitory Activity on SNARE Complex Formation

Neurotransmitter release from presynaptic nerve terminals is regulated by SNARE complex-mediated synaptic vesicle fusion. Tomosyn, a negative regulator of neurotransmitter release, which is composed of N-terminal WD40 repeats, a tail domain, and a C-terminal VAMP-like domain, is known to inhibit SNARE complex formation by sequestering target SNAREs (t-SNAREs) upon interaction of its C-terminal VAMP-like domain with t-SNAREs. However, it remains unclear how the inhibitory activity of tomosyn is regulated. Here we show that the tail domain functions as a regulator of the inhibitory activity of tomosyn through intramolecular interactions. The binding of the tail domain to the C-terminal VAMP-like domain interfered with the interaction of the C-terminal VAMP-like domain with t-SNAREs, and thereby repressed the inhibitory activity of tomosyn on the SNARE complex formation. The repressed inhibitory activity of tomosyn was restored by the binding of the tail domain to the N-terminal WD40 repeats. These results indicate that the probable conformational change of tomosyn mediated by the intramolecular interactions of the tail domain controls its inhibitory activity on the SNARE complex formation, leading to a regulated inhibition of neurotransmitter release.Synaptic vesicles are transported to the presynaptic plasma membrane where Ca²⁺ channels are located. Depolarization induces Ca²⁺ influx into the cytosol of nerve terminals through the Ca²⁺ channels, and this Ca²⁺ influx initiates the fusion of the vesicles with the plasma membrane, finally leading to exocytosis of neurotransmitters (1). Soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP)² receptors (SNAREs) are essential for synaptic vesicle exocytosis (2-5). Synaptic vesicles are endowed with vesicle-associated membrane protein 2 (VAMP-2) as a vesicular SNARE, whereas the presynaptic plasma membrane is endowed with syntaxin-1 and SNAP-25 as target SNAREs. VAMP-2 interacts with SNAP-25 and syntaxin-1 to form a stable SNARE complex (6-9). The formation of the SNARE complex then brings synaptic vesicles and the plasma membrane into close apposition, and provides the energy that drives the mixing of the two lipid bilayers (3-5, 9).Tomosyn is a syntaxin-1-binding protein that we originally identified (10). Tomosyn contains N-terminal WD40 repeats, a tail domain, and a C-terminal domain homologous to VAMP-2. The C-terminal VAMP-like domain (VLD) of tomosyn acts as a SNARE domain that competes with VAMP-2. Indeed, a structural study of the VLD revealed that the VLD, syntaxin-1, and SNAP-25 assemble into a SNARE complex-like structure (referred to as tomosyn complex hereafter) (11). Tomosyn inhibits SNARE complex formation by sequestering t-SNAREs through the tomosyn complex formation, and thereby inhibits SNARE-dependent neurotransmitter release. The large N-terminal region of tomosyn shares similarity to the Drosophila tumor suppressor lethal giant larvae (Lgl), the mammalian homologues M-Lgl1 and M-Lgl2, and yeast proteins Sro7p and Sro77p (12, 13). Consistent with the function of tomosyn, Lgl family members play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane in yeast and epithelial cells (12, 13). However, only tomosyn, Sro7, and Sro77 have the tail domains and the VLDs, suggesting that their structural regulation is evolutionally conserved. Recently, the crystal structure of Sro7 was solved and revealed that the tail domain of Sro7 binds its WD40 repeats (14). Sec9, a yeast counterpart of SNAP-25, also binds the WD40 repeats of Sro7. This binding inhibits the SNARE complex formation and exocytosis by sequestering Sec9. In addition, binding of the tail domain to the WD40 repeats causes a conformational change of Sro7 and prevents the interaction of the WD40 repeats with Sec9, leading to regulation of the inhibitory activity of Sro7 on the SNARE complex formation (14). However, the solved structure of Sro7 lacks its VLD. Therefore, involvement of the activity of the VLD in the conformational change of Sro7 remains elusive.Genetic studies in Caenorhabditis elegans showed that TOM-1, an ortholog of vertebrate tomosyn, inhibits the priming of synaptic vesicles, and that this priming is modulated by the balance between TOM-1 and UNC-13 (15, 16). Tomosyn was also shown to be involved in inhibition of the exocytosis of dense core granules in adrenal chromaffin cells and PC12 cells (17, 18). Thus, evidence is accumulating that tomosyn acts as a negative regulator for formation of the SNARE complex, thereby inhibiting various vesicle fusion events. However, the precise molecular mechanism regulating the inhibitory action of tomosyn has yet to be elucidated.In the present study, we show that the tail domain of tomosyn binds both the WD40 repeats and the VLD and functions as a regulator for the inhibitory activity of tomosyn on the SNARE complex formation. Our results indicate that the probable conformational change of tomosyn mediated by the intramolecular interactions of the tail domain serves for controlling the inhibitory activity of the VLD.     

F-Actin Dynamics in Neurospora crassa

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.Actins are highly conserved proteins found in all eukaryotes and have an enormous variety of cellular roles. The monomeric form (globular actin, or G-actin) can self-assemble, with the aid of numerous actin-binding proteins (ABPs), into microfilaments (filamentous actin, or F-actin), which, together with microtubules, form the two major components of the fungal cytoskeleton. Numerous pharmacological and genetic studies of fungi have demonstrated crucial roles for F-actin in cell polarity, exocytosis, endocytosis, cytokinesis, and organelle movement (6, 7, 20, 34, 35, 51, 52, 59). Phalloidin staining, immunofluorescent labeling, and fluorescent-protein (FP)-based live-cell imaging have revealed three distinct subpopulations of F-actin-containing structures in fungi: patches, cables, and rings (1, 14, 28, 34, 60, 63, 64). Actin patches are associated with the plasma membrane and represent an accumulation of F-actin around endocytic vesicles (3, 26, 57). Actin cables are bundles of actin filaments stabilized with cross-linking proteins, such as tropomyosins and fimbrin, and are assembled by formins at sites of active growth, where they form tracks for myosin V-dependent polarized secretion and organelle transport (10, 16, 17, 27, 38, 47, 48). Cables, unlike patches, are absolutely required for polarized growth in the budding yeast Saccharomyces cerevisiae (34, 38). Contractile actomyosin rings are essential for cytokinesis in budding yeast, whereas in filamentous fungi, actin rings are less well studied but are known to be involved in septum formation (20, 28, 34, 39, 40).Actin cables and patches have been particularly well studied in budding yeast. However, there are likely to be important differences between F-actin architecture and dynamics in budding yeast and those in filamentous fungi, as budding yeasts display only a short period of polarized growth during bud formation, which is followed by isotropic growth over the bud surface (10). Sustained polarized growth during hyphal morphogenesis is a defining feature of filamentous fungi (21), making them attractive models for studying the roles of the actin cytoskeleton in cell polarization, tip growth, and organelle transport.In Neurospora crassa and other filamentous fungi, disruption of the actin cytoskeleton leads to rapid tip swelling, which indicates perturbation of polarized tip growth, demonstrating a critical role for F-actin in targeted secretion to particular sites on the plasma membrane (7, 22, 29, 56). Immunofluorescence studies of N. crassa have shown that F-actin localizes to hyphal tips as “clouds” and “plaques” (7, 54, 59). However, immunolabeling has failed to reveal actin cables in N. crassa and offers limited insights into F-actin dynamics. Live-cell imaging of F-actin architecture and dynamics has not been accomplished in N. crassa, yet it is expected to yield key insights into cell polarization, tip growth, and intracellular transport.We took advantage of a recently developed live-cell imaging probe for F-actin called Lifeact (43). Lifeact is a 17-amino-acid peptide derived from the N terminus of the budding yeast actin-binding protein Abp140 (5, 63) and has recently been demonstrated to be a universal live-cell imaging marker for F-actin in eukaryotes (43). Here, we report the successful application of fluorescent Lifeact fusion constructs for live-cell imaging of F-actin in N. crassa. We constructed two synthetic genes consisting of Lifeact fused to “synthetic” green fluorescent protein (sGFP) (S65T) (henceforth termed GFP) (12) or red fluorescent protein (TagRFP) (33) and expressed these constructs in various N. crassa strains. In all strain backgrounds, fluorescent Lifeact constructs clearly labeled actin patches, cables, and rings and revealed a direct association of F-actin structures with sites of cell polarization and active tip growth. Our results demonstrate the efficacy of Lifeact as a nontoxic live-cell imaging probe in N. crassa.     

A Thermodynamic Model of Microtubule Assembly and Disassembly

Microtubules are self-assembling polymers whose dynamics are essential for thenormal function of cellular processes including chromosome separation andcytokinesis. Therefore understanding what factors effect microtubule growth isfundamental to our understanding of the control of microtubule based processes.An important factor that determines the status of a microtubule, whether it isgrowing or shrinking, is the length of the GTP tubulin microtubule cap. Here, wederive a Monte Carlo model of the assembly and disassembly of microtubules. Weuse thermodynamic laws to reduce the number of parameters of our model and, inparticular, we take into account the contribution of water to the entropy of thesystem. We fit all parameters of the model from published experimental datausing the GTP tubulin dimer attachment rate and the lateral and longitudinalbinding energies of GTP and GDP tubulin dimers at both ends. Also we calculateand incorporate the GTP hydrolysis rate. We have applied our model and can mimicpublished experimental data, which formerly suggested a single layer GTP tubulindimer microtubule cap, to show that these data demonstrate that the GTP cap canfluctuate and can be several microns long.     

MTB-3, a Microtubule Plus-End Tracking Protein (+TIP) of Neurospora crassa

The microtubule (MT) “plus end” constitutes the platform for the accumulation of a structurally and functionally diverse group of proteins, collectively called “MT plus-end tracking proteins” (+TIPs). +TIPs control MT dynamics and link MTs to diverse sub-cellular structures. Neurospora crassa MicroTubule Binding protein-3 (MTB-3) is the homolog of yeast EB1, a highly conserved +TIP. To address the function of MTB-3, we examined strains with mtb-3 deletions, and we tagged MTB-3 with GFP to assess its dynamic behavior. MTB-3-GFP was present as comet-like structures distributed more or less homogeneously within the hyphal cytoplasm, and moving mainly towards the apex at speeds up to 4× faster than the normal hyphal elongation rates. MTB-3-GFP comets were present in all developmental stages, but were most abundant in mature hyphae. MTB-3-GFP comets were observed moving in anterograde and retrograde direction along the hypha. Retrograde movement was also observed as originating from the apical dome. The integrity of the microtubular cytoskeleton affects the presence and dynamics of MTB-3-GFP comets, while actin does not seem to play a role. The size of MTB-3-GFP comets is affected by the absence of dynactin and conventional kinesin. We detected no obvious morphological phenotypes in Δmtb-3 mutants but there were fewer MTs in Δmtb-3, MTs were less bundled and less organized. Compared to WT, both MT polymerization and depolymerization rates were significantly decreased in Δmtb-3. In summary, the lack of MTB-3 affects overall growth and morphological phenotypes of N. crassa only slightly, but deletion of mtb-3 has strong effect on MT dynamics.     

Functional Interactions between Mldp (LSDP5) and Abhd5 in the Control of Intracellular Lipid Accumulation

Cellular lipid metabolism is regulated in part by protein-protein interactions near the surface of intracellular lipid droplets. This work investigated functional interactions between Abhd5, a protein activator of the lipase Atgl, and Mldp, a lipid droplet scaffold protein that is highly expressed in oxidative tissues. Abhd5 was highly targeted to individual lipid droplets containing Mldp in microdissected cardiac muscle fibers. Mldp bound Abhd5 in transfected fibroblasts and directed it to lipid droplets in proportion to Mldp concentration. Analysis of protein-protein interactions in situ demonstrated that the interaction of Abhd5 and Mldp occurs mainly, if not exclusively, on the surface of lipid droplets. Oleic acid treatment rapidly increased the interaction between Abhd5 and Mldp, and this effect was suppressed by pharmacological inhibition of triglyceride synthesis. The functional role of the Abhd5-Mldp interaction was explored using a mutant of mouse Abhd5 (E262K) that has greatly reduced binding to Mldp. Mldp promoted the subcellular colocalization and interaction of Atgl with wild type, but not mutant, Abhd5. This differential interaction was reflected in cellular assays of Atgl activity. In the absence of Mldp, wild type and mutant Abhd5 were equally effective in reducing lipid droplet formation. In contrast, mutant Abhd5 was unable to prevent lipid droplet accumulation in cells expressing Mldp despite considerable targeting of Atgl to lipid droplets containing Mldp. These results indicate that the interaction between Abhd5 and Mldp is dynamic and essential for regulating the activity of Atgl at lipid droplets containing Mldp.Growing evidence indicates that lipogenesis and lipolysis are regulated by protein-protein interactions that occur on the surface of specialized intracellular lipid droplets (1, 2). PAT³ (perilipin, adipophilin, and TIP-47) proteins, are thought to be key regulators of these processes by serving as scaffolds that organize and regulate the protein trafficking at lipid droplet surfaces (1–3). Mldp (muscle lipid droplet protein; alternatively, OXPAT, LSDP5) is a PAT family member that is highly expressed in tissues, like muscle and liver, having high oxidative capacity (4–6). Expression of Mldp is up-regulated under conditions such as fasting and diabetes, in which the systemic supply of lipid to target tissues is increased, and in vitro studies suggest that Mldp plays a role in facilitating triglyceride storage as well as fatty acid oxidation (4–6). It is not presently known how Mldp is involved in these functions, but we hypothesize that it is likely to involve direct or indirect interactions with lipases and lipase co-activators (3, 7).Abhd5 (α/β hydrolase domain-containing protein 5; alternatively CGI-58) is an evolutionarily conserved protein that acts as a potent activator of Atgl (adipose triglyceride lipase; alternatively, PNPLA2, desnutrin, TTS-2.1) (8). Both proteins are expressed in a variety of tissues, and rare homozygous mutations of either gene in humans produces a similar (but not identical) lipid storage disease that is characterized by ectopic lipid accumulation in skin, muscle, and liver (9–11). Regulation of lipid metabolism by Abhd5 is not fully understood. Abhd5 has been shown to bind perilipin (Plin) (12, 13), and it has been proposed that the phosphorylation-dependent release of Abhd5 is a means of initiating lipolysis via activation of Atgl (3, 7). Abhd5 is expressed in several tissues that lack Plin (12), raising the possibility that this co-activator might interact with additional PAT proteins.In the experiments detailed below, we investigated the potential interaction of Mldp and Abhd5 in vivo and in vitro. Our results show that Mldp and Abhd5 interact in vivo and in vitro. This interaction occurs on the surface of intracellular lipid droplets and is promoted by triglyceride synthesis. Atgl and Mldp are targeted to the same lipid droplets, and the interaction of Abhd5 with Mldp appears to be critical for regulating Atgl activity at these droplets.     

The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila

Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (stai) gene in the maintenance of axonal microtubules and regulation of axonal transport in Drosophila . The stai gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of stai function in Drosophila , which has a single stai gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the stai gene family. This has allowed us to identify an essential function for stai in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of stai mutant Drosophila , we also observe additional neurological phenotypes associated with loss of stai function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in stai deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of Drosophila mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that stai has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport.     

Host Cell Invasion by Toxoplasma gondii Is Temporally Regulated by the Host Microtubule Cytoskeleton

Toxoplasma gondii is an obligate intracellular protozoan parasite that invades and replicates within most nucleated cells of warm-blooded animals. The basis for this wide host cell tropism is unknown but could be because parasites invade host cells using distinct pathways and/or repertoires of host factors. Using synchronized parasite invasion assays, we found that host microtubule disruption significantly reduces parasite invasion into host cells early after stimulating parasite invasion but not at later time points. Host microtubules are specifically associated with the moving junction, which is the site of contact between the host cell and the invading parasite. Host microtubules are specifically associated with the moving junction of those parasites invading early after stimulating invasion but not with those invading later. Disruption of host microtubules has no effect on parasite contact, attachment, motility, or rate of penetration. Rather, host microtubules hasten the time before parasites commence invasion. This effect on parasite invasion is distinct from the role that host microtubules play in bacterial and viral infections, where they function to traffic the pathogen or pathogen-derived material from the host cell''s periphery to its interior. These data indicate that the host microtubule cytoskeleton is a structure used by Toxoplasma to rapidly infect its host cell and highlight a novel function for host microtubules in microbial pathogenesis.Toxoplasma gondii is an obligate intracellular protozoan parasite that is capable of causing disease in fetuses and immunocompromised individuals (23). The parasite infects a wide range of nucleated cells of most warm-blooded animals. The mechanisms underlying this wide tropism are not known but could be due to either the parasite infecting cells using a ubiquitously expressed host receptor and associated machinery, inserting its own receptor into the host cell''s plasma membrane, or using multiple host cell receptors/machinery (5).Toxoplasma invasion is a multistep, complex process consisting of parasite contact to host cells, intimate attachment, parasite motility, and then penetration (5). Host cell contact is a loose, low-affinity interaction that is mediated by parasite surface antigens. An unknown signal then triggers the release of proteins from a specialized secretory organelle called micronemes whose contents include proteins that function as adhesins. This is then followed by parasite gliding motility on the host cell surface. At some point, proteins from a second secretory organelle, named rhoptries, are exocytosed. Among these rhoptry proteins, several (RON2, RON4, RON5, and RON8) are part of a preformed complex that binds the previously secreted AMA1 microneme protein (1, 2, 20, 33). Together, these proteins form the moving junction complex, which defines the parasite entry site on the host cell plasma membrane. Parasite penetration occurs by the parasite propelling itself forward, via acto-myosin-dependent motility, into the host plasma membrane (35). This causes an invagination of the plasma membrane resulting in the formation of the parasitophorous vacuole (PV), which is the compartment that the parasite resides in throughout its time in the host cell. However, host plasma membrane-associated proteins are selectively incorporated into the developing PV such that glycosylphosphatidylinositol (GPI)-linked proteins are included, while single-pass transmembrane proteins are excluded (7, 24).In contrast to parasite molecules that function during invasion, few host cell components involved in this process are known. A notable exception is the finding that host Arp2/3-dependent actin polymerization promotes Toxoplasma invasion (11). Nevertheless, how actin or other host molecules function during invasion remains to be determined. The host microtubule cytoskeleton has been widely studied for its role during receptor-mediated endocytosis, as well as in bacterial and viral infections, where microtubules act to facilitate cargo transport from the host cell periphery to the interior (8, 15, 27, 29, 40). Consistent with this role in cargo transport, host microtubules also promote trafficking of rhoptry proteins secreted into the host cell (12). However, whether this host cell structure functions during parasite invasion per se is unknown.Here, we tested the hypothesis that host microtubules are used by Toxoplasma tachyzoites to penetrate into its host cell. Using synchronized parasite invasion assays, we find that disruption of host microtubules significantly reduces parasite invasion into host cells early after stimulating parasite invasion but not at later time points. Host microtubules are localized to the moving junction but, unlike their previously described role in pathogen invasion, host microtubules promote tachyzoite invasion by hastening the time that parasites initiate invasion.     

Interactions Among Selected Endoparasitic Nematodes and Three Pseudomonads on Alfalfa

Meloidogyne hapla, Pratylenchus penetrans, and Helicotylenchus dihystera, reduced the growth of ''Saranac AR alfalfa seedlings when applied at concentrations of 50 nematodes per plant. All except P. penetrans reduced seedling growth when applied at 25 per seedling. M. hapla reduced growth when applied at 12 per seedling. Nematodes interacted with three pseudomonads to produce greater growth reductions than were obtained with single pathogens, suggesting synergistic relationships. Ditylenchus dipsaci, applied at 25 or 50 nematodes per seedling, reduced plant weight compared with weights of control plants, but did not interact with test bacteria. All of the nematodes except D. dipsaci produced root wounds which were invaded by bacteria.     

Single Neuron Ubiquitin-Proteasome Dynamics Accompanying Inclusion Body Formation in Huntington Disease

The accumulation of mutant protein in intracellular aggregates is a common feature of neurodegenerative disease. In Huntington disease, mutant huntingtin leads to inclusion body (IB) formation and neuronal toxicity. Impairment of the ubiquitin-proteasome system (UPS) has been implicated in IB formation and Huntington disease pathogenesis. However, IBs form asynchronously in only a subset of cells with mutant huntingtin, and the relationship between IB formation and UPS function has been difficult to elucidate. Here, we applied single-cell longitudinal acquisition and analysis to monitor mutant huntingtin IB formation, UPS function, and neuronal toxicity. We found that proteasome inhibition is toxic to striatal neurons in a dose-dependent fashion. Before IB formation, the UPS is more impaired in neurons that go on to form IBs than in those that do not. After forming IBs, impairment is lower in neurons with IBs than in those without. These findings suggest IBs are a protective cellular response to mutant protein mediated in part by improving intracellular protein degradation.Huntington disease (HD)⁴ is a progressive incurable neurodegenerative disorder caused by the expansion of a polyglutamine (polyQ) stretch in the N-terminal end of the huntingtin (htt) protein above a threshold length of ∼36 (1). The deposition of polyQ-expanded aggregated mutant htt in inclusion bodies (IBs) is a hallmark of HD, and IBs are found in human post-mortem samples, transgenic mouse brain, and cell-culture models (2). The accumulation of ubiquitinated proteins in IBs has implicated the ubiquitin-proteasome system (UPS) in the pathogenesis of HD, amyotrophic lateral sclerosis, Parkinson disease, and polyQ-mediated disorders (3).The UPS is a major pathway of intracellular protein degradation. After a series of three reactions, each catalyzed by a different set of enzymes, ubiquitin, a 76-amino acid polypeptide, forms an isopeptide bond with the amino group of lysine residues on substrate proteins. Several lysine residues within ubiquitin are sites for more ubiquitin additions. Once a protein accumulates four or more ubiquitins, it is efficiently targeted to the proteasome for degradation. The proteasome binds polyubiquitinated substrates and hydrolyzes ubiquitin isopeptide bonds, releasing ubiquitin moieties before degrading substrate proteins through chymotrypsin-like, trypsin-like, and post-glutamyl peptidase activities (3).Increased polyubiquitin levels and changes in ubiquitin linkages accompany the accumulation of UPS substrates in the brains of HD patients and transgenic mice and in cellular HD models (4). UPS substrates accumulate throughout the cell in polyQ models, even before IB formation (5, 6). This has added to the confusion over whether polyQ expansion leads to toxicity through direct impairment of proteasomal degradation. Proteasomes have been reported to cleave polyQ stretches efficiently (7), inefficiently (8), or essentially not at all (9). In vivo, polyQ-dependent degeneration occurs with no detectable proteasome inhibition (10, 11) or is tightly linked to it (12, 13). The inability of some studies to detect UPS impairment in HD models may be due to the limited sensitivity of conventional approaches to identify cell-to-cell variations in UPS function.The relationship between IB formation and UPS function has been difficult to determine. Protein turnover in cells with IBs is evidently reduced and accompanied by the accumulation of cellular proteins (14–16); HEK293 cells containing mutant htt IBs have a greater degree of UPS impairment than those without IBs (5). Proteasome subunits and heat shock proteins colocalize with IBs, but it is unclear if this colocalization facilitates protein delivery or unfolding at the mouth of active proteasomes, or if it harms proteasome function by sequestering essential cellular machinery (18). Some IBs are relatively static (8, 25), but the proteins in others are dynamically exchanged with cytoplasmic and nuclear pools (19, 20).UPS function is critical to cellular homeostasis. Deletion of one of the two inducible polyubiquitin genes in mice leads to lower intracellular ubiquitin levels in germ cells and hypothalamic neurons. These same populations undergo cell-cycle arrest and hypothalamic neurodegeneration, respectively (22, 23). Cell lines expressing mutant huntingtin accumulate ubiquitinated proteins and undergo cell-cycle arrest in G2/M (5). In neurons, UPS impairment may lead to cell death through an accumulation of signals for apoptosis, a decrease in NF-κB signaling, sensitization to other toxic stimuli, remodeling of synapses, retraction of neurites, or other unidentified mechanisms (24). The effect of UPS impairment depends on cell type and cell cycle, and the relationship between UPS impairment and striatal neuronal survival is largely unknown.Diffuse species of mutant htt induce IB formation and neuronal death in a protein concentration-dependent manner (2). IB formation delays neuronal death, suggesting that IB formation helps neurons cope with toxic diffuse mutant htt. Whether the effect of IB formation on survival is mediated through UPS function has been difficult to determine. IB formation and neuronal death occur asynchronously in overlapping but distinct subsets of neurons that express mutant htt. The observation that IB formation is not required for UPS impairment also complicates population analysis (6, 26).To explore this problem, we applied single-cell analysis. We tracked single neurons over their entire lifetimes, gaining spatial and temporal resolution while simultaneously monitoring IB formation, UPS inhibition, and neuronal toxicity.     

Kinesin-3 Responds to Local Microtubule Dynamics to Target Synaptic Cargo Delivery to the Presynapse

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Extending the Microtubule/Microfibril Paradigm : Cellulose Synthesis Is Required for Normal Cortical Microtubule Alignment in Elongating Cells

The cortical microtubule array provides spatial information to the cellulose-synthesizing machinery within the plasma membrane of elongating cells. Until now data indicated that information is transferred from organized cortical microtubules to the cellulose-synthesizing complex, which results in the deposition of ordered cellulosic walls. How cortical microtubules become aligned is unclear. The literature indicates that biophysical forces, transmitted by the organized cellulose component of the cell wall, provide a spatial cue to orient cortical microtubules. This hypothesis was tested on tobacco (Nicotiana tabacum L.) protoplasts and suspension-cultured cells treated with the cellulose synthesis inhibitor isoxaben. Isoxaben (0.25–2.5 μm) inhibited the synthesis of cellulose microfibrils (detected by staining with 1 μg mL⁻¹ fluorescent dye and polarized birefringence), the cells failed to elongate, and the cortical microtubules failed to become organized. The affects of isoxaben were reversible, and after its removal microtubules reorganized and cells elongated. Isoxaben did not depolymerize microtubules in vivo or inhibit the polymerization of tubulin in vitro. These data are consistent with the hypothesis that cellulose microfibrils, and hence cell elongation, are involved in providing spatial cues for cortical microtubule organization. These results compel us to extend the microtubule/microfibril paradigm to include the bidirectional flow of information.     

Lymphocyte Activation Gene 3 (LAG-3) Modulates the Ability of CD4 T-cells to Be Suppressed In Vivo

Lymphocyte Activation Gene – 3 (LAG-3) is an immune checkpoint molecule that regulates both T-cell activation and homeostasis. However, the molecular mechanisms underlying LAG-3’s function are generally unknown. Using a model in which LAG-3 blockade or absence reliably augmented homeostatic proliferation in vivo, we found that IL-2 and STAT5 are critical for LAG-3 function. Similarly, LAG-3 blockade was ineffective in the absence of regulatory T-cells (Treg), suggesting an important role for LAG-3 in either the responsiveness of conventional T-cells (Tconv) to regulation, or a relative defect in the ability of LAG-3 KO regulatory T-cells (Treg) to suppress the proliferation of Tconv. In this model, LAG-3 KO Treg suppressed proliferation in a manner fairly similar to wild-type (WT) Treg, but LAG-3 KO Tconv were relatively resistant to suppression. Further studies also identified a role for LAG-3 in the induction/expansion of Treg. Finally, we found that LAG-3 blockade (or knockout) led to a relative skewing of naïve CD4 T-cells toward a T_H1 phenotype both in vitro and in in vivo. Together, these data suggest that LAG-3 expression on Tconv cells makes them more susceptible to Treg based suppression, and also regulates the development of a T_H1 T-cell response.