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The UL34 gene product of herpes simplex virus type 2 is a tail-anchored type II membrane protein that is significant for virus envelopment.

The UL34 gene of herpes simplex virus type 2 (HSV-2) is highly conserved in the herpesvirus family. The UL34 gene product was identified In lysates of HSV-2-infected cells as protein species with molecular masses of 31 and 32.5 kDa, the latter being a phosphorylated product. Synthesis of these proteins occurred at late times post-infection and was highly dependent on viral DNA synthesis. Immunofluorescence assays revealed that the UL34 protein was localized in the cytoplasm in a continuous net-like structure, closely resembling the staining pattern of the endoplasmic reticulum (ER), in both HSV-2-infected cells and in cells transiently expressing UL34 protein. Deletion mutant analysis showed that this colocalization required the C terminus of the UL34 protein. The UL34 protein associated with virions but not with A, B or C capsids. We treated virions, HSV-2-infected cells and cells expressing the UL34 protein with a protease in order to examine the topology of the UL34 protein. In addition, we constructed UL34 deletion mutant proteins and examined their intracellular localization. Our data strongly support the hypothesis that the UL34 protein is inserted into the viral envelope as a tail-anchored type II membrane protein and is significant for virus envelopment.

Animals↗

Multiple insertional events, restricted by the genetic background, have led to acquisition of pathogenicity island IIJ96-like domains among Escherichia coli strains of different clinical origins.

We investigated the dissemination of pathogenicity island (PAI) II(J96)-like elements (hra, hly, cnf1, and pap) among 455 Escherichia coli isolates from children and adults with urinary tract infection (UTI), neonates with meningitis or colonized healthy neonates, and 74 reference strains by means of PCR phylogenetic grouping, ribotyping, and PCR analysis of virulence genes. Colocalization of these genes was documented by pulsed-field gel electrophoresis followed by Southern hybridization and long-range PCR (LRPCR) between the hra and the papG alleles. Site-specific insertion of the PAI was determined by LRPCR between hra and tRNA flanking sequences. hra, hly, and cnf1 were found in 113 isolates and consistently colocalized, constituting the backbone of PAI II(J96)-like domains. The prevalence of PAI II(J96)-like domains was significantly higher among UTI isolates than among neonatal meningitis and commensal isolates. These domains were restricted to a few ribotypes of group B2. In contrast to the consistent colocalization of hra, hly, and cnf1, the pap operon was varied: 12% of strains exhibited an allelic exchange of the papG class III allele (papGIII) for the papG class II allele (papGII) (only UTI isolates), and the pap operon was deleted in 23% of strains. No strains harbored papGIII outside the PAI, which appears to be the only source of this allele. PAI II(J96)-like domains were inserted in the vicinities of three different tRNAs--pheU (54%), leuX (29%), and pheV (15%)--depending on the genetic backgrounds and origins of the isolates. Multiple insertional events restricted by the genetic background have thus led to PAI II(J96) acquisition. Specific genetic backgrounds and insertion sites may have played a role in additional recombination processes for E. coli adaptation to different ecological niches.

Adhesins, Escherichia coli↗

Endocytosis is a critical step in entry of subgroup B avian leukosis viruses.

The avian leukosis virus (ALV) entry mechanism is controversial, with evidence for and against a low-pH requirement for viral fusion. To further address this question, we tested the entry of human immunodeficiency virus type 1 (HIV-1) pseudotyped with the envelope protein of subgroup B ALV (ALV-B) in the presence of three different lysosomotropic agents. These lysosomotropic agents were able to block the entry of wild-type and pseudotyped ALV-B in two different cell lines, strongly suggesting that ALV-B requires a low-pH step for entry. ALV-B and pH-dependent Semliki Forest virus (SFV) entered cells with slower uptake kinetics than HIV-1, which is pH independent. These slow uptake rates support the theory that ALV-B utilizes endocytic pathways to enter cells. Using immunofluorescence and electron microscopy analysis, we visualized the colocalization of virus particles with the endosomal marker transferrin and demonstrated virus particles in clathrin-coated vesicles and endosome-like structures. Surprisingly, a low-pH treatment did not overcome the inhibition of ALV-B entry by lysosomotropic agents. This indicates that, in contrast to SFV, ALV-B is unable to fuse at the cellular surface, even at a low pH. Taken together, our findings suggest that endocytosis and a subsequent low-pH step are critical for successful ALV-B infection.

Ammonium Chloride↗

Ca2+ content and expression of an acidocalcisomal calcium pump are elevated in intracellular forms of Trypanosoma cruzi.

The survival of a eukaryotic protozoan as an obligate parasite in the interior of a eukaryotic host cell implies its adaptation to an environment with a very different ionic composition from that of its extracellular habitat. This is particularly important in the case of Ca2+, the intracellular concentration of which is 3 orders of magnitude lower than the extracellular value. Ca2+ entry across the plasma membrane is a widely recognized mechanism for Ca2+ signaling, needed for a number of intracellular processes, and obviously, it would be restricted in the case of intracellular parasites. Here we show that Trypanosoma cruzi amastigotes possess a higher Ca2+ content than the extracellular stages of the parasite. This correlates with the higher expression of a calcium pump, the gene for which was cloned and sequenced. The deduced protein product (Tca1) of this gene has a calculated molecular mass of 121,141 Da and exhibits 34 to 38% identity with vacuolar Ca2+-ATPases of Saccharomyces cerevisiae and Dictyostelium discoideum, respectively. The tca1 gene suppresses the Ca2+ hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca2+ accumulation. Indirect immunofluorescence and immunoelectron microscopy analysis indicate that Tca1 colocalizes with the vacuolar H+-ATPase to the plasma membrane and to intracellular vacuoles of T. cruzi. These vacuoles were shown to have the same size and distribution as the calcium-containing vacuoles identified by the potassium pyroantimoniate-osmium technique and as the electron-dense vacuoles observed in whole unfixed parasites by transmission electron microscopy and identified in a previous work (D. A. Scott, R. Docampo, J. A. Dvorak, S. Shi, and R. D. Leapman, J. Biol. Chem. 272:28020-28029, 1997) as being acidic and possessing a high calcium content (i.e., acidocalcisomes). Together, these results suggest that acidocalcisomes are distinct from other previously recognized organelles present in these parasites and underscore the ability of intracellular parasites to adapt to the hostile environment of their hosts.

Amino Acid Sequence↗

IQGAP1 mediates VE-cadherin-based cell-cell contacts and VEGF signaling at adherence junctions linked to angiogenesis.

OBJECTIVE: Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating reactive oxygen species (ROS) production primarily through the VEGF receptor-2 (VEGFR2). One of the initial responses in established vessels to stimulate angiogenesis is loss of vascular endothelial (VE)-cadherin-based cell-cell adhesions; however, little is known about the underlying mechanisms. IQGAP1 is a novel VEGFR2 binding protein, and it interacts directly with actin, cadherin, and beta-catenin, thereby regulating cell motility and morphogenesis. METHODS AND RESULTS: Confocal microscopy analysis shows that IQGAP1 colocalizes with VE-cadherin at cell-cell contacts in unstimulated human endothelial cells (ECs). VEGF stimulation reduces staining of IQGAP1 and VE-cadherin at the adherens junction without affecting interaction of these proteins. Knockdown of IQGAP1 using siRNA inhibits localization of VE-cadherin at cell-cell contacts, VEGF-stimulated recruitment of VEGFR2 to the VE-cadherin/beta-catenin complex, ROS-dependent tyrosine phosphorylation of VE-cadherin, which is required for loss of cell-cell contacts and capillary tube formation. IQGAP1 expression is increased in a mouse hindlimb ischemia model of angiogenesis. CONCLUSIONS: IQGAP1 is required for establishment of cell-cell contacts in quiescent ECs. To induce angiogenesis, it may function to link VEGFR2 to the VE-cadherin containing adherens junctions, thereby promoting VEGF-stimulated, ROS-dependent tyrosine phosphorylation of VE-cadherin and loss of cell-cell contacts.

Adherens Junctions↗

Bone marrow-derived cells do not incorporate into the adult growing vasculature.

Bone marrow-Derived cells have been proposed to form new vessels or at least incorporate into growing vessels in adult organisms under certain physiological and pathological conditions. We investigated whether bone marrow-Derived cells incorporate into vessels using mouse models of hindlimb ischemia (arteriogenesis and angiogenesis) and tumor growth. C57BL/6 wild-type mice were lethally irradiated and transplanted with bone marrow cells from littermates expressing enhanced green fluorescent protein (GFP). At least 6 weeks after bone marrow transplantation, the animals underwent unilateral femoral artery occlusions with or without pretreatment with vascular endothelial growth factor or were subcutaneously implanted with methylcholanthrene-induced fibrosarcoma (BFS-1) cells. Seven and 21 days after surgery, proximal hindlimb muscles with growing collateral arteries and ischemic gastrocnemius muscles as well as grown tumors and various organs were excised for histological analysis. We failed to colocalize GFP signals with endothelial or smooth muscle cell markers. Occasionally, the use of high-power laser scanning confocal microscopy uncovered false-positive results because of overlap of different fluorescent signals from adjacent cells. Nevertheless, we observed accumulations of GFP-positive cells around growing collateral arteries (3-fold increase versus nonoccluded side, P<0.001) and in ischemic distal hindlimbs. These cells were identified as fibroblasts, pericytes, and primarily leukocytes that stained positive for several growth factors and chemokines. Our findings suggest that in the adult organism, bone marrow-Derived cells do not promote vascular growth by incorporating into vessel walls but may function as supporting cells.

Animals↗

ZO-1: lamellipodial localization in a corneal fibroblast wound model.

PURPOSE: To explore the roles of ZO-1 in corneal fibroblasts and myofibroblasts in a model of wounding. METHODS: Antibodies were used to identify ZO-1 in cultured rabbit corneal fibroblasts by immunocytochemistry, Western blot analysis, and immunoprecipitation. For colocalization studies, antibodies to beta-catenin, cadherins, connexins, integrins, alpha-actinin, and cortactin were used. G- and F-actin were identified by DNase and rhodamine phalloidin, respectively. To study ZO-1 localization during cell migration, confluent corneal fibroblasts were subjected to scrape-wounding and evaluated by immunocytochemistry. RESULTS: As predicted from previous studies, ZO-1 colocalized with cadherins and connexin 43 in intercellular junctions. The study revealed a new finding: ZO-1 was also detected at the leading edge of lamellipodia, especially in motile wounded fibroblasts and in freshly plated fibroblasts, before the formation of cell-cell contacts. In fibroblast lysates, ZO-1 largely partitioned to the detergent-soluble fraction compared with myofibroblast lysates, indicating that much of the fibroblast ZO-1 is not associated with insoluble structural components. Lamellipodial ZO-1 colocalized with G-actin, alpha-actinin, and cortactin, which are proteins involved with actin remodeling and cell migration. Integrins alpha5beta1 and alphavbeta3 also localized to the leading edge of migrating fibroblasts, and the association of ZO-1 with integrin was confirmed by immunoprecipitation. Finally, alkaline phosphatase treatment of fibroblast lysate decreased the molecular mass of ZO-1 in lysates of cells grown in serum, demonstrating that, in activated fibroblasts, ZO-1 is phosphorylated. CONCLUSIONS: ZO-1's appearance at the leading edge of migrating fibroblasts makes it a candidate for a role in the initiation and organization of integrin-dependent fibroblast adhesion complexes formed during migration and adhesion. Further, phosphorylation of ZO-1 may regulate its cellular localization.

Animals↗

Homo- and heteromeric assembly of TRPV channel subunits.

The vanilloid receptor-related TRP channels (TRPV1-6) mediate thermosensation, pain perception and epithelial Ca(2+) entry. As the specificity of TRPV channel heteromerization and determinants governing the assembly of TRPV subunits were largely elusive, we investigated the TRPV homo- and heteromultimerization. To analyze the assembly of TRPV subunits in living cells, we generated fluorescent fusion proteins or FLAG-tagged TRPV channel subunits. The interaction between TRPV subunits was assessed by analysis of the subcellular colocalization, fluorescence resonance energy transfer and coimmunoprecipitation. Our results demonstrate that TRPV channel subunits do not combine arbitrarily. With the exception of TRPV5 and TRPV6, TRPV channel subunits preferentially assemble into homomeric complexes. Truncation of TRPV1, expression of cytosolic termini of TRPV1 or TRPV4 and construction of chimeric TRPV channel subunits revealed that the specificity and the affinity of the subunit interaction is synergistically provided by interaction modules located in the transmembrane domains and in the cytosolic termini. The relative contribution of intramolecularly linked interaction modules presumably controls the overall affinity and the specificity of TRPV channel assembly.

Animals↗

The maize aberrant pollen transmission 1 gene is a SABRE/KIP homolog required for pollen tube growth.

Maize (Zea mays) pollen tubes grow in the styles at a rate of >1 microm/sec. We describe here a gene required to attain that striking rate. The aberrant pollen transmission 1 (apt1) gene of maize was identified by an Ac-tagged mutation that displayed a severe pollen transmission deficit in heterozygotes. Rare apt1 homozygotes can be recovered, aided by phenotypic selection for Ac homozygotes. Half of the pollen in heterozygotes and most of the pollen in homozygotes germinate short and twisted pollen tubes. The apt1 gene is 26 kb long, makes an 8.6-kb pollen-specific transcript spliced from 22 exons, and encodes a protein of 2607 amino acids. The APT1 protein is homologous to SABRE and KIP, Arabidopsis proteins of unknown function involved in the elongation of root cortex cells and pollen tubes, respectively. Subcellular localization analysis demonstrates that APT1 colocalizes with a Golgi protein marker in growing tobacco pollen tubes. We hypothesize that the APT1 protein is involved in membrane trafficking and is required for the high secretory demands of tip growth in pollen tubes. The apt1-m1(Ac) mutable allele is an excellent tool for selecting Ac transpositions because of the strong negative selection pressure operating against the parental Ac site.

Arabidopsis Proteins↗

A high endothelial venule secretory protein, mac25/angiomodulin, interacts with multiple high endothelial venule-associated molecules including chemokines.

We previously reported that mac25/angiomodulin (AGM), a 30-kDa secretory protein, is abundantly expressed in high endothelial venules (HEVs), which play a crucial role in lymphocyte trafficking to the lymph nodes and Peyer's patches. We report that mac25/AGM interacts preferentially with certain molecules that are expressed in or around HEVs. In particular, mac25/AGM interacted with not only the extracellular matrix proteins and glycosaminoglycans that are expressed in most blood vessels including HEVs, but also with some chemokines that are implicated in the regulation of lymphocyte trafficking, such as the secondary lymphoid-tissue chemokine (SLC; CCL21), IFN-gamma-inducible protein 10 (IP-10; CXCL10), and RANTES (CCL5). The binding of mac25/AGM to SLC and IP-10 was dose-dependent and saturable. The binding to IP-10 could be inhibited by SLC but not by a non-mac25/AGM-binding chemokine, EBI1-ligand chemokine (ELC; CCL19). Interestingly, mac25/AGM failed to interact with 18 other chemokines, suggesting that it binds to certain chemokines preferentially. Immunohistochemical analysis indicated that mac25/AGM colocalizes at least partially with SLC and IP-10 at the basal lamina of HEVs. Upon binding with mac25/AGM, SLC and IP-10 retained all their Ca(2+)-signaling activity in vitro, suggesting that mac25/AGM can hold and present chemokines in the basal lamina of HEVs. These results imply that mac25/AGM plays a multifunctional role, serving not only as an adhesion protein to interact with glycosaminoglycans and extracellular matrix proteins but also as a molecule to present chemokines so that lymphocytes extravasating through HEVs receive further directional cues subsequent to the luminal encounter with lymphoid chemokines.

Amino Acid Sequence↗

Identification of the region in the N-terminal domain responsible for the cytoplasmic localization of Myoc/Tigr and its association with microtubules.

Mutations in the MYOC/TIGR gene are associated with juvenile open-angle glaucoma and in some cases may be involved in the formation of sporadic primary open-angle glaucoma in humans. To better understand the functions of the MYOC/TIGR protein, its intracellular distribution was investigated using green fluorescent protein (GFP) as a marker. The results indicated that the recombinant mouse and human Myoc/Tigr-GFP proteins are located in the cytoplasm of the transfected cells in which they colocalize with microtubules. Deletion analysis demonstrated that the N-terminal region (positions 1-124 and 15-138 in the mouse and human proteins, respectively) encoded by exon 1 is critical for the cytoplasmic localization of Myoc/Tigr. Most of the known mutations in the human MYOC/TIGR gene implicated in juvenile and sporadic primary open-angle glaucoma formation are located outside the region responsible for the cytoplasmic localization of the protein. However, some of these mutations may alter the tertiary structure of the protein and subsequently modify its interaction with microtubules.

Age of Onset↗

Alterations of ocular nitric oxide synthase in human glaucoma.

PURPOSE: The authors recently reported that sites of outflow resistance and regulation in the human eye are highly enriched in the endothelial isoform of nitric oxide (NO) synthase (ecNOS). In vasculature, ecNOS activation is associated with altered vascular resistance and, in the stomach, defects in NO are associated with pathologic gastric hypertension. Because human glaucoma sometimes is associated with an increase in intraocular pressure and resistance changes in the aqueous outflow pathway (OP), the authors have investigated the possibility that alterations in NO or defects in NO-synthesizing tissues might exist in glaucomatous eyes. METHODS: Occurrence, distribution, and extent of sites of ocular NO production in the anterior segments of 16 normal eyes (10 patients) and 17 eyes (12 patients) with a history of primary open-angle glaucoma (POAG) were determined using the NO-indicator marker, NADPH-diaphorase (NADPH-d), which is known to colocalize with ecNOS immunoreactivity. Analysis of NADPH-d reactivity in tissues was combined with examination of overall cell distribution and use of neuron-specific markers. RESULTS: The ciliary muscle (CM) and OP of glaucomatous eyes showed marked differences in the amount and distribution of NADPH-d and alterations in gross structure. NADPH-d reactivity was decreased in trabecular meshwork (TM) and Schlemm's canal, and there was a marked reduction of anterior longitudinal CM fibers that insert near (and may normally regulate resistance in) the TM. CONCLUSION: Abnormalities in NO or NO-containing cells occur in POAG. These abnormalities may be causally related to glaucoma or may be a manifestation of the disease or its treatment. In either case, such alterations, together with recent pharmacologic studies showing that NO-mimicking nitrovasodilators alter IOP, indicate that NO has relevance to the course, treatment, or both, or some forms of this disease.

Aged↗

The Armadillo family protein p0071 is a VE-cadherin- and desmoplakin-binding protein.

p0071, a member of the armadillo protein family, localizes to both adherens junctions and desmosomes in epithelial cells and exhibits homology to the adherens junction protein p120 and the desmosomal protein plakophilin-1. p0071 is also present at dermal microvascular endothelial intercellular junctions and colocalizes with VE-cadherin, an endothelium-specific cadherin that associates with both actin and intermediate filament networks. To define the role of p0071 in junction assembly, p0071 was tested for interactions with other components of the endothelial junctional complex. In transient expression assays, p0071 colocalized with and formed complexes with both VE-cadherin and desmoplakin. Deletion analysis using the yeast two-hybrid system revealed that the armadillo repeat domain of p0071 bound directly to VE-cadherin. Site-directed mutagenesis experiments demonstrated that p0071 and p120 bound to the same region on the cytoplasmic tail of VE-cadherin and that overexpression of p0071 could displace p120 from intercellular junctions. In contrast to VE-cadherin, desmoplakin was found to associate with the non-armadillo head domain of p0071. Cotransfections and triple-label immunofluorescence analysis revealed that VE-cadherin colocalization with desmoplakin in transfected COS cells required p0071, suggesting that p0071 may couple VE-cadherin to desmoplakin. Based on previous findings that both VE-cadherin and desmoplakin play central roles in vasculogenesis, these new results suggest that p0071 may play an important role in endothelial junction assembly and in the morphogenic events associated with vascular remodeling.

Adherens Junctions↗

A quantitative determination of multi-protein interactions by the analysis of confocal images using a pixel-by-pixel assessment algorithm.

MOTIVATION: Recent advances in confocal microscopy have allowed scientists to assess the expression, and to some extent, the interaction/colocalization of multiple molecules within cells and tissues. In some instances, accurately quantifying the colocalization of two or more proteins may be critical. This can require the acquisition of multiple Z plane images (Z stacks) throughout a specimen and, as such, we report here the successful development of a freeware, open-source image analysis tool, IMAJIN_COLOC, developed in PERL (v. 5.8, build 806), using the PERLMagick libraries (ImageMagick). Using a pixel-by-pixel analysis algorithm, IMAJIN_COLOC can analyze images for antigen expression (any number of colors) and can measure all possible combinations of colocalization for up to three colors by analyzing a Z stack gallery acquired for each sample. The simultaneous (i.e. in a single pass) analysis of three-color colocalization, and batch analysis capabilities are distinctive features of this program. RESULTS: A control image, containing known individual and colocalized pixel counts, was used to validate the accuracy of IMAJIN_COLOC. As further validation, pixel counts and colocalization values from the control image were compared to those obtained with the software packaged with the Zeiss laser-scanning microscope (LSM AIM, version 3.2). The values from both programs were found to be identical. To demonstrate the applicability of this program in addressing novel biological questions, we examined the role of neurons in eliciting an immune reaction in response to viral infection. Specifically, we successfully examined expression of the chemokine RANTES in measles virus (MV) infected hippocampal neurons and quantified changes in RANTES production throughout the disease period. The resultant quantitative data were also evaluated visually, using a gif image created during the analysis. AVAILABILITY: PERL (ActivePerl, version 5.8) is available at activestate.com; the PERLMagick libraries are available at imagemagick.org, and IMAJIN_COLOC, the source code and user documentation can be downloaded from http://www.fda.gov/cber/research/imaging/imageanalysis.htm.

Algorithms↗

Vacuolar invertase regulates elongation of Arabidopsis thaliana roots as revealed by QTL and mutant analysis.

The possible role of the sucrose-splitting enzymes sucrose synthase and invertase in elongating roots and hypocotyls of Arabidopsis was tested by using a combination of histochemical methods and quantitative trait locus (QTL) analysis. Lengths of roots and hypocotyls correlated better with invertase activities than with sucrose synthase activities. The highest correlations were observed with activities in the elongating zones of roots. The genetic basis of these correlations was studied by using QTL analysis. Several loci, affecting invertase activity, colocated with loci that had an effect on root or hypocotyl length. Further fine mapping of a major locus for root length, but not for hypocotyl length (top chromosome 1), consistently showed colocation with the locus for invertase activity containing a gene coding for a vacuolar invertase. The analysis of a functional knockout line confirmed the role of this invertase in root elongation, whereas other invertase genes might play a role in hypocotyl elongation. Thus, we show the power of QTL analysis, combined for morphological and biochemical traits, followed by fine-mapping and mutant analysis, in unraveling the function of genes and their role in growth and development.

Arabidopsis↗

Further studies on the endocytic activity of Tritrichomonas foetus.

The endocytic activity of Tritrichomonas foetus was studied at the ultrastructural level using gold-labeled macromolecules (bovine lactoferrin, human and bovine transferrin, bovine albumin, human low-density lipoprotein, horseradish peroxidase, and protein A). All macromolecules were ingested by the protozoan. Binding experiments showed that only bovine lactoferrin bound to the parasite surface in a process that could be inhibited by the unlabeled protein, suggesting that it binds and is internalized via receptors. Label-fracture experiments showed that the receptors were distributed in clusters that did not colocalize with intramembranous particles. Kinetics analysis of the internalization of bovine lactoferrin and horseradish peroxidase, associated with the cytochemical detection of acid phosphatase, revealed that proteins were rapidly ingested through small uncoated vesicles and delivered to acid phosphatase-containing compartments. The colocalization of gold-labeled proteins and reaction product indicative of enzyme activity was confirmed by electron spectroscopic imaging. Simultaneous incubation of cells in the presence of two proteins labeled with gold particles of different diameters showed that they were ingested through the same pathway and were concentrated into cytoplasmic vacuoles corresponding to lysosome-like organelles. These data suggest that the endocytic process in T. foetus is very rapid and that the intracellular pathway for receptor-mediated and fluid-phase endocytosis seems to be the same.

Animals↗

Human Bub1: a putative spindle checkpoint kinase closely linked to cell proliferation.

Eukaryotic cells have evolved a mechanism that delays the onset of anaphase until chromosomes are properly positioned on the spindle. To understand the molecular basis of such surveillance mechanism in human cells, we have cloned a full-length cDNA encoding a putative mitotic checkpoint kinase termed hBub1. Sequence comparison reveals that hBub1 is a structurally conserved protein, sharing 23% amino acid residue identity with BUB1 of budding yeast. In addition, the NH2-terminal portion (161 amino acids) of hBub1 shows a significant homology to yeast MAD3, a protein also known to be involved in the mitotic checkpoint response pathway. Northern blot analyses show that the hBub1 mRNA level is abundantly expressed in tissues or cells with a high mitotic index. When Dami cells undergo terminal differentiation after treatment with phorbol ester, hBub1 expression in this cell line is down-regulated rapidly. The hBub1 protein level is low in G1 and remains relatively constant in S, G2, and M phases. Immunofluorescence analysis shows that hBub1 protein colocalizes with a centromere-kinetochore antigen CREST in interphase, mitotic prophase, and nocodazole-treated cells. Antibody electroporation experiments show that hBub1 is an important component of the spindle checkpoint pathway. Furthermore, fluorescence in situ hybridization analysis maps the hBub1 gene to chromosome 2q12-13. Our studies suggest that hBub1 expression is restricted to proliferating cells and appears to be involved in regulating cell cycle progression. The molecular cloning of hBub1 cDNA will facilitate the study of its role in spindle checkpoint control as well as its potential role in certain genetic disorders.

Amino Acid Sequence↗

Cell-surface MuSK self-association: a crucial role for the putative signal sequence.

The receptor tyrosine kinase MuSK plays a crucial role-both as a signaling molecule and structurally-in the process of clustering nicotinic acetylcholine receptors at the neuromuscular junction. Immunofluorescence microscopy of transiently transfected fibroblasts has been used to visualize the cell-surface distribution of MuSK, which is found in discrete, punctate clusters. This distribution does not result from targeting of MuSK to identified plasma membrane subdomains, and MuSK's association with itself is specific, as MuSK clusters at the cell surface are segregated from clusters of other cotransfected receptor tyrosine kinases. A mutational analysis, using coexpressed pairs of MuSK mutants and chimeras, demonstrates that the putative signal peptide is both necessary and sufficient for association with MuSK. Removal of the intracellular domain or most of the extracellular domain, or replacement of the transmembrane domain, does not abolish MuSK self-association. The N-terminus of the MuSK protein, however, is sufficient to recruit another receptor tyrosine kinase to MuSK clusters. Quantitation and statistical analysis of the amount of colocalization between coexpressed MuSK mutants and chimeras confirm these results.

Amino Acid Sequence↗