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PubMed1996

Dynamic changes in nuclear architecture during mitosis: on the role of protein phosphorylation in spindle assembly and chromosome segregation.

During mitosis, the vertebrate cell nucleus undergoes profound changes in architecture. At the onset of mitosis, the nuclear envelope breaks down, the nuclear lamina is depolymerized, and interphase chromatin is condensed to chromosomes. Concomitantly, cytoplasmic microtubules are reorganized into a mitotic spindle apparatus, a highly dynamic structure required for the segregation of sister chromatids. Many of the above events are controlled by reversible phosphorylation. Hence, our laboratory is interested in characterizing the kinases involved in promoting progression through mitosis and in identifying their relevant substrates. Prominent among the kinases responsible for regulating entry into mitosis is the Cdc2 kinase, the first member of the cyclin dependent kinase (Cdk) family. Recently, we found that Cdc2 phosphorylates HsEg5, a human kinesin-related motor protein associated with centrosomes and the spindle apparatus. Our results indicate that phosphorylation regulates the association of HsEg5 with the mitotic spindle and that the function of this plus-end directed motor is essential for centrosome separation and bipolar spindle formation. Another kinase implicated in regulating progression through mitosis is Plk1 (polo-like kinase 1), the human homologue of the Drosophila gene product "polo." By antibody microinjection we have found that Plk1 is required for the functional maturation of centrosomes and hence for entry into mitosis. Furthermore, we found that microinjected anti-Plk1 antibodies caused a more severe block to cell cycle progression in diploid fibroblasts than in immortalized tumor cells. This observation hints at the existence of a checkpoint linking Cdc2 activation to the presence of functional centrosomes.

Animals↗
PubMed2002

Human TPX2 is required for targeting Aurora-A kinase to the spindle.

Aurora-A is a serine-threonine kinase implicated in the assembly and maintenance of the mitotic spindle. Here we show that human Aurora-A binds to TPX2, a prominent component of the spindle apparatus. TPX2 was identified by mass spectrometry as a major protein coimmunoprecipitating specifically with Aurora-A from mitotic HeLa cell extracts. Conversely, Aurora-A could be detected in TPX2 immunoprecipitates. This indicates that subpopulations of these two proteins undergo complex formation in vivo. Binding studies demonstrated that the NH2 terminus of TPX2 can directly interact with the COOH-terminal catalytic domain of Aurora-A. Although kinase activity was not required for this interaction, TPX2 was readily phosphorylated by Aurora-A. Upon siRNA-mediated elimination of TPX2 from cells, the association of Aurora-A with the spindle microtubules was abolished, although its association with spindle poles was unaffected. Conversely, depletion of Aurora-A by siRNA had no detectable influence on the localization of TPX2. We propose that human TPX2 is required for targeting Aurora-A kinase to the spindle apparatus. In turn, Aurora-A might regulate the function of TPX2 during spindle assembly.

Aurora Kinases↗
PubMed1999

Analysis of Huntingtin-associated protein 1 in mouse brain and immortalized striatal neurons.

Huntingtin, the protein product of the Huntington's disease (HD) gene, is expressed with an expanded polyglutamine domain in the brain and in nonneuronal tissues in patients with HD. Huntingtin-associated protein 1 (HAP-1), a brain-enriched protein, interacts preferentially with mutant huntingtin and thus may be important in HD pathogenesis. The function of HAP-1 is unknown, but recent evidence supports a role in microtubule-dependent organelle transport. We examined the subcellular localization of HAP-1 with an antibody made against the NH2-terminus of the protein. In immunoblot assays of mouse brain and immortalized striatal neurons, HAP-1 subtypes A and B migrated together at about 68 kD and separately at 95 kD and 110 kD, respectively. In dividing clonal striatal cells, HAP-1 localized to the mitotic spindle apparatus, especially at spindle poles and on vesicles and microtubules of the spindle body. Postmitotic striatal neurons had punctate HAP-1 labeling throughout the cytoplasm. Western blot analysis of protein extracts obtained after subcellular fractionation and differential centrifugation of the clonal striatal cells showed that HAP-1B was preferentially enriched in membrane fractions. Electron microscopic study of adult mouse basal forebrain and striatum showed HAP-1 localized to membrane-bound organelles including large endosomes, tubulovesicular structures, and budding vesicles in neurons. HAP-1 was also strongly associated with an unusual large "dense" organelle. Microtubules were labeled in dendrites and axonal fibers. Results support a role for HAP-1 in vesicle trafficking and organelle movement in mitotic cells and differentiated neurons and implicate HAP-1B as the predominant molecular subtype associated with vesicle membranes in striatal neurons.

Animals↗
PubMed1987

The location and orientation of mitotic figures during histogenesis of the rat cerebellar cortex.

The location and orientation of mitotic spindle fibers in the developing cerebellar cortex was examined in rats. In both the transverse and sagittal planes, the mitotic spindle apparatus of cells in the superficial region (EGL-S) was generally oriented perpendicular to the pial surface, whereas that of cells in the deeper aspects (EGL-D) was oriented parallel. The mitotic frequency of cells in the EGL-S was 2- to 6-fold greater than was that in the EGL-D. The mitotic spindle arrangement of dividing cells located adjacent to Purkinje neurons, and presumed to be Bergmann glia, was always perpendicular to the pial surface. Autoradiographic studies revealed DNA synthesis in both the EGL-S and EGL-D. Mitotic cells do not migrate from the EGL-S to the EGL-D, as evidenced by disruption of the cytoskeletal matrix with vinblastine sulfate. Our results suggest that the orientation of the spindle apparatus may play an important role in the emergence of the precise geometry characteristic of the adult cerebellum, and could signify that the phenotypic fate of neural cell precursors is determined prior to the onset of overt differentiation.

Animals↗
PubMed2002

CP248, a derivative of exisulind, causes growth inhibition, mitotic arrest, and abnormalities in microtubule polymerization in glioma cells.

Exisulind (sulindac sulfone) and two potent derivatives, CP248 and CP461, have been shown previously to cause growth inhibition and apoptosis in several types of human carcinoma cell lines. These and related compounds have not been previously studied with respect to glioma cell lines. In the present study, we found that these three compounds caused marked growth inhibition in four rat glioma and eight human glioma cell lines, with IC50 values of 150, 1, and 0.075 microm, respectively. When studied at these concentrations exisulind and CP461 had no significant effect on the cell cycle profile of glioma cells, but CP248 caused marked arrest in mitosis. Detailed studies of CP248 in the 9L rat gliosarcoma cell line indicated that treatment with 0.075 microM CP248 caused abnormalities in the spindle apparatus and activation of the spindle assembly check point. In interphase glioma cells, CP248 stabilized microtubules (MTs) at low concentrations (0.075 microM) and depolymerized MTs at higher concentrations (0.2-0.4 microM). In NIH 3T3 fibroblasts, 0.1 microM CP248 caused extensive MT depolymerization. CP248 also caused MT depolymerization when added to assembled MTs in vitro, which indicated that it can directly affect MTs, perhaps because it shares certain structural similarities with Colcemid. In glioma cells, the effects of CP248 on MTs were independent of the previously reported effects of this compound on activation of protein kinase G. Therefore, CP248 is a novel MT-active agent that may be useful in the treatment of glioblastoma, and possibly other types of cancer, because of its dual effects on protein kinase G and MTs.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Photodynamic effects of the cationic porphyrin, mesotetra(4N-methylpyridyl)porphine, on microtubules of HeLa cells.

The treatment of HeLa human carcinoma cells with mesotetra(4N-methylpyridyl)porphine (T4MPyP) and blue light led to damage of the microtubules (MTs). The morphologies of interphase MTs and the mitotic spindle apparatus were analysed by immunofluorescence staining of alpha-tubulin. The extent of MT damage depended on the light dose and the time after photodynamic treatment. After a period of 1 h after irradiation with doses of 0.3 or 1.5 J cm-2 (sublethal conditions, corresponding to survival rates of 90% and 60% respectively), the normal MT network arrangement of interphase cells and the mitotic spindle apparatus of many cells were clearly affected. However, these effects were found to be transient, and several hours after irradiation most MTs resumed control morphology. Higher irradiation doses (4.5 J cm-2, lethal conditions, less than 10% cell survival) resulted in the irreversible alteration of interphase and mitotic MTs. The change in MT organization appeared to be the reason for the observed increase in the mitotic index (MI) after sublethal doses. The largest increase in MI was detected 6 h after treatment (twofold increase over untreated cells) for both sublethal light doses. Most of the cells in mitosis corresponded to metaphase, the number of ana-telophase cells being greatly reduced for the first hours after irradiation with a dose of 1.5 J cm-2. The results, which resemble those observed with inhibitors of MT assembly, suggest that MTs might represent an important target for T4MPyP action.

Fluorescent Antibody Technique↗
PubMed2000

The proteasome is involved in the first metaphase-to-anaphase transition of meiosis in rat oocytes.

The proteasome engages in protein degradation as a regulatory process in biological transactions. Among other cellular processes, the proteasome participates in degradation of ubiquinated cyclins in mitosis. However, its role in meiosis has not been established. Resumption of meiosis in the oocyte involves the activation of maturation promoting factor (MPF), a complex of p34cdc2 and cyclin B. Inactivation of this factor, occurring between the two meiotic divisions, is associated with degradation of cyclin B. In this study, we examined the possible involvement of the proteasome in regulation of the exit from metaphase I in spontaneously maturing rat oocytes. We found that upon resumption of meiosis, proteasomes translocate to the spindle apparatus. We further demonstrated that specific inhibitors of proteasome catalytic activity, MG132 and lactacystin, blocked polar body extrusion. Chromosome and microtubule fluorescent staining verified that MG132-treated oocytes were arrested at metaphase I. Intervention of proteasomal action with this inhibitor also resulted in accumulation of cyclin B and elevated activity of MPF. These data demonstrate that proteasomal catalytic activity is absolutely essential for the decrease in MPF activity and completion of the first meiotic division. Its translocation to the spindle apparatus may facilitate the timely degradation of cyclin B.

Acetylcysteine↗
PubMed1992

Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, before the onset of anaphase, the spindle apparatus is always positioned with one spindle pole at, or through, the neck between the mother cell and the growing bud. This spindle orientation enables proper chromosome segregation to occur during anaphase, allowing one replicated genome to be segregated into the bud and the other to remain in the mother cell. In this study, we synchronized a population of cells before the onset of anaphase such that > 90% of the cells in the population had spindles with the correct orientation, and then disrupted specific cytoskeletal elements using temperature-sensitive mutations. Disruption of either the astral microtubules or actin function resulted in improper spindle orientation in approximately 40-50% of the cells. When cells with disrupted astral microtubules or actin function entered into anaphase, there was a 100-200-fold increase in the frequency of binucleated cell bodies. Thus, the maintenance of proper spindle orientation by these cytoskeletal elements was essential for proper chromosome segregation. These data are consistent with the model that proper spindle orientation is maintained by directly or indirectly tethering the astral microtubules to the actin cytoskeleton. After nuclear migration, but before anaphase, bulk chromosome movement occurs within the nucleus apparently because the chromosomes are attached to a mobile spindle. The frequency and magnitude of bulk chromosome movement is greatly diminished by disruption of the astral microtubules but not by disruption of the nonkinetochore spindle microtubules. These results suggest that astral microtubules are not only important for spindle orientation before anaphase, but they also mediate force on the spindle, generating spindle displacement and in turn chromosome movement. Potential roles for this force in spindle assembly and orientation are discussed.

Actins↗
PubMed1997

Disruption of microtubule assembly and spindle formation as a mechanism for the induction of aneuploid cells by sodium arsenite and vanadium pentoxide.

Arsenic and vanadium are important environmental and industrial pollutants. Due to their widespread occurrence and potential genotoxicity, we studied the aneuploidy-inducing effects of these elements in cultured human lymphocytes using a variety of techniques including fluorescence in situ hybridization (FISH) with DNA probes for chromosomes 1 and 7, immunostaining of the lymphocyte spindle apparatus, and an in vitro assay measuring the polymerization and depolymerization of tubulin. Dose-related increases in hyperdiploidy were seen in lymphocyte cultures treated with sodium arsenite (NaAsO2) or vanadium pentoxide (V2O5) over concentrations ranging from 0.001 to 0.1 microM. NaAsO2-treated cells from different donors exhibited similar hyperdiploid frequencies, whereas substantial inter-individual variability was seen in the V2O5-treated cells. Examination of the spindle apparatus using an anti-beta-tubulin antibody indicated that these compounds might disrupt spindle formation by interacting with microtubules. Additional in vitro assays using purified tubulin indicated that both compounds inhibited microtubule assembly and induced tubulin depolymerization. These results indicate that in vitro exposure to both NaAsO2 and V2O5 can induce aneuploidy in human lymphocytes, and that this effect may occur through a disruption of microtubule function.

Aneuploidy↗
PubMed1995

Mitotic disturbances and micronucleus induction in Syrian hamster embryo fibroblast cells caused by asbestos fibers.

Asbestos and other mineral fibers have long been known to induce lung cancer and mesothelioma. However, the primary mechanisms of fiber-induced carcinogenesis still remain unclear. We investigated the occurrence of mitotic disturbances induced by asbestos (amosite, crocidolite, chrysotile) in an in vitro approach using Syrian hamster embryo (SHE) fibroblast cells. The following endpoints were investigated: micronucleus formation as a result of mitotic disturbances and characterization of the induced micronucleus population by kinetochore staining and visualization of the spindle apparatus. Supravital UV-microscopy was used to analyze changes in interphase chromatin structure, impaired chromatid separation, and blocked cytokinesis. All three asbestos fiber types induced a high frequency of micronucleus formation in SHE cells (> 200/2000 cells) in a dose-dependent manner (0.1-5.0 micrograms/cm2), with a maximum between 48 hr and 66 hr exposure time. At higher concentrations (more than 5.0 micrograms/cm2) the micronucleus formation decreased again as a result of increased toxicity. Kinetochore staining of micronuclei revealed that 48 +/- 2% of asbestos-induced micronuclei reacted positively with CREST (antikinetochore) serum. Furthermore, spindle apparatus deformations occurred in cells with disturbed metaphases and anaphases, while the spindle fiber morphology appeared unchanged. Our results show that asbestos fibers may cause both loss and breakage of chromosomes in the absence of direct interaction with spindle fibers.

Animals↗
PubMed1999

Characterization of novel F-actin envelopes surrounding nuclei during cleavage of a polychaete worm.

F-actin accumulations and their possible functions were investigated during cleavage of the polychaete Ophryotrocha puerilis. Unusual cytoplasmic accumulations of F-actin were detected which have never been described before in animal embryos. As shown by TRITC-phalloidin labeling, envelopes of F-actin surrounded late prophase nuclei for a short period of time. DTAF-immunofluorescence of beta-tubulin showed that the F-actin envelope was closely associated with microtubules of the developing spindle apparatus. However, experimental disassembly of microtubules by nocodazole did not prevent the assembly of the F-actin envelope. Disturbance of F-actin envelope formation by cytochalasin B did not alter the course of mitotic events, i.e. position of the nuclei and orientation of the spindle apparatus were not affected, although the respective blastomeres remained uncleaved. However, disassembly of the F-actin envelope correlated temporally with breakdown of the nuclear envelope. Therefore, it is suggested that this new structure plays a role in fragmentation of the nuclear envelope during cleavage of Ophryotrocha puerilis.

Actins↗
PubMed2005

Subcellular localization of nardilysin during mouse oocyte maturation.

We have previously shown that the peptidase, nardilysin, contains a bipartite nuclear localization signal that permits the enzyme to cycle between the nucleus and cytoplasm. In the present study, we report that nardilysin accumulates in the nucleus of an oocyte as a function of its maturation. Nardilysin is predominantly localized in the cytoplasm of an oocyte when initially placed into culture. The enzyme starts to accumulate in the nucleus within 30 min of in vitro culture. After 3 h, nardilysin is found as a spherical structure surrounded by condensed chromosomal DNA. After 18 h of in vitro culture, it co-localizes with beta-tubulin at the spindle apparatus. Cilostamide, a phosphodiesterase 3A inhibitor that inhibits meiosis, blocks accumulation of nuclear nardilysin. This finding demonstrates that the nuclear entry of nardilysin is tightly controlled in the oocyte. Taken together, these experiments strongly suggest a role for nardilysin in meiosis through its dynamic translocation from cytosol to nucleus, and then to the spindle apparatus.

Active Transport, Cell Nucleus↗
PubMed1988

1,6-Dinitropyrene causes spindle disturbances and chromosomal damage in V79 Chinese hamster cells.

We have investigated the cytogenetic effect of 1,6-dinitropyrene (1,6-DNP) in Chinese hamster V79 cells. The chemical caused a dose-dependent increase in the incidence of initial and full C-mitoses, polyploid mitoses, ana-telophases with lagging chromosomes, non-disjunction and multipolar configurations, in a range of 0.05-5 microM. These findings indicate that 1,6-DNP interferes with the functioning of the spindle apparatus in V79 cells. Early signs of spindle disturbances were seen at 1,6-DNP concentrations which only moderately reduced cell growth and division. Analysis of structural chromosomal aberrations revealed the appearance of chromatid-type aberrations with open breaks and exchanges accompanied by gaps. The results indicate that 1,6-DNP is both a spindle-disturbing and a clastogenic agent in V79 cells.

Animals↗
PubMed2006

In vitro induction of micronuclei by monofunctional methanesulphonic acid esters: possible role of alkylation mechanisms.

Six monofunctional alkylating methanesulphonates of widely varying structures were investigated in the in vitro micronucleus assay with Syrian hamster embryo fibroblast cells. The results were compared with the alkylating activities measured in the 4-(nitrobenzyl)pyridine test (NBP-test) and the N-methyl mercaptoimidazole (MMI-test) as measures for S(N)2 reactivity as well as in the triflouoroacetic acid (TFA) solvolysis and the hydrolysis reaction as measures for S(N)1 reactivity in order to provide insights into the role of alkylation mechanisms on induction of micronuclei. Moreover we compared the results of micronucleus assay with those of the Ames tests in strain TA 100 and TA1535 and with those of the SOS chromotest with the strains PQ37, PQ243, PM21 and GC 4798. The potency of methanesulphonates to induce micronuclei depended only to a certain degree, on the total alkylating activity (S(N)1 and S(N)2 reactivity). An inverse, significant correlation between the Ames test and the micronucleus assay was observed and an inverse correlation between the micronucleus assay and the SOS chromotest with the different strains. The results indicate that the primary mechanism leading to induction of micronuclei is not O-alkylation in DNA as it is the case in the Ames test with the hisG46 strains TA1535 and TA100 and not N-alkylation as with the SOS chromotest. There is evidence that protein alkylation, e.g. in the spindle apparatus in mitosis is decisive for induction of micronuclei by alkylating compounds. The structurally voluminous methanesulphonates 2-phenyl ethyl methanesulphonate and 1-phenyl-2-propyl methanesulphonate show a clear higher micronuclei inducing potency than the other tested though the bulky methanesulphonates possess a lower total alkylating activity than the others. This effect can be explained by a higher disturbance during mitosis after alkylation of the spindle apparatus with the structurally more bulky methanesulphonates.

Alkylating Agents↗
PubMed2006

Rae1 interaction with NuMA is required for bipolar spindle formation.

In eukaryotic cells, the faithful segregation of daughter chromosomes during cell division depends on formation of a microtubule (MT)-based bipolar spindle apparatus. The Nuclear Mitotic Apparatus protein (NuMA) is recruited from interphase nuclei to spindle MTs during mitosis. The carboxy terminal domain of NuMA binds MTs, allowing a NuMA dimer to function as a "divalent" crosslinker that bundles MTs. The messenger RNA export factor, Rae1, also binds to MTs. Lowering Rae1 or increasing NuMA levels in cells results in spindle abnormalities. We have identified a mitotic-specific interaction between Rae1 and NuMA and have explored the relationship between Rae1 and NuMA in spindle formation. We have mapped a specific binding site for Rae1 on NuMA that would convert a NuMA dimer to a "tetravalent" crosslinker of MTs. In mitosis, reducing Rae1 or increasing NuMA concentration would be expected to alter the valency of NuMA toward MTs; the "density" of NuMA-MT crosslinks in these conditions would be diminished, even though a threshold number of crosslinks sufficient to stabilize aberrant multipolar spindles may form. Consistent with this interpretation, we found that coupling NuMA overexpression to Rae1 overexpression or coupling Rae1 depletion to NuMA depletion prevented the formation of aberrant spindles. Likewise, we found that overexpression of the specific Rae1-binding domain of NuMA in HeLa cells led to aberrant spindle formation. These data point to the Rae1-NuMA interaction as a critical element for normal spindle formation in mitosis.

Antigens, Nuclear↗
PubMed2005

Clathrin is required for the function of the mitotic spindle.

Clathrin has an established function in the generation of vesicles that transfer membrane and proteins around the cell. The formation of clathrin-coated vesicles occurs continuously in non-dividing cells, but is shut down during mitosis, when clathrin concentrates at the spindle apparatus. Here, we show that clathrin stabilizes fibres of the mitotic spindle to aid congression of chromosomes. Clathrin bound to the spindle directly by the amino-terminal domain of clathrin heavy chain. Depletion of clathrin heavy chain using RNA interference prolonged mitosis; kinetochore fibres were destabilized, leading to defective congression of chromosomes to the metaphase plate and persistent activation of the spindle checkpoint. Normal mitosis was rescued by clathrin triskelia but not the N-terminal domain of clathrin heavy chain, indicating that stabilization of kinetochore fibres was dependent on the unique structure of clathrin. The importance of clathrin for normal mitosis may be relevant to understanding human cancers that involve gene fusions of clathrin heavy chain.

Animals↗
PubMed1994

Nuclear mitotic apparatus protein (NuMA): spindle association, nuclear targeting and differential subcellular localization of various NuMA isoforms.

We have recently shown that the nuclear mitotic apparatus protein (NuMA) is composed of at least three isoforms that differ mainly at the carboxy terminus, and are generated by alternative splicing of a common mRNA precursor from a single NuMA gene (J. Cell Sci. (1993) 104, 249-260). Transient expression of human NuMA-1 isoform (T33/p230) in Chinese hamster ovary polyoma (CHOP) cells showed that NuMA-1 was present in interphase nuclei and was concentrated at the polar regions of the spindle apparatus in mitotic cells. However, expression of two other isoforms (NuMA-m and -s) revealed a distinct subcellular localization. NuMA-m (U4/p195) and NuMA-s (U6/p194) were present in the interphase cytosol and appeared to be mainly located at the centrosomal region. When cells entered into mitosis, however, NuMA-m and -s moved to the mitotic spindle pole. Analysis of a series of linker scanning-mutants and NuMA/beta-galactosidase chimeric proteins showed that residues 1972-2007 of NuMA-1 constitute a novel nuclear localization signal (NLS) and residues 1538-2115 are necessary and sufficient for spindle association. Further analysis of the NLS by site-specific mutagenesis indicated that Lys1988 is essential for nuclear targeting, whereas Arg1984 is not. These results have allowed us tentatively to assign specific biological activities to distinct structural domains of the NuMA polypeptide.

Amino Acid Sequence↗
PubMed1996

The spindle kinesin-like protein HsEg5 is an autoantigen in systemic lupus erythematosus.

OBJECTIVE: Autoantibodies directed against the mitotic spindle apparatus (MSA) have been shown to target an antigen referred to as NuMA (nuclear mitotic apparatus). In this study, we identified a second MSA antigen as the spindle kinesin-like protein HsEg5. We studied the frequency of antibodies to HsEg5 in human sera that demonstrate the MSA pattern of staining, the frequency of autoantibodies to HsEg5 in patients with systemic lupus erythematosus (SLE), and the clinical features of patients with antibodies to HsEg5. METHODS: A prototype serum from an SLE patient was used to isolate a 4.8-kilobase complementary DNA (cDNA) from a HeLa cDNA library. Western blot, immunoprecipitation, and sequence analysis revealed that the antigen was an approximately 130-kd protein, HsEg5. The frequency of autoantibodies to recombinant HsEg5 in 51 sera that demonstrated an MSA pattern of staining on HEp-2 and HeLa cells was detected by immunoblotting 2 constructs of the cDNA. The clinical features of patients with antibodies directed against HsEg5 was obtained by retrospective chart review. RESULTS: The antigen responsible for the MSA-35 pattern was identified as the human kinesin-like protein HsEg5. Seven of 51 sera (14%) that demonstrated an MSA pattern of staining reacted with recombinant HsEg5. Six of 7 of the HsEg5-positive patients (86%) had SLE, and 1 had Sjögren's syndrome. The indirect immunofluorescent staining pattern of sera that reacted with HsEg5 could be distinguished from the other sera that reacted with NuMA. In an unselected cohort of 52 SLE patients, 3 (6%) had autoantibodies reactive with the recombinant HsEg5. CONCLUSION: Autoantibodies to MSA fall into 2 major classes: those reactive with NuMA and those reactive with HsEg5. Autoantibodies to HsEg5 are found in a lower frequency than NuMA in sera that demonstrate the MSA pattern of staining and appear to be specifically associated with SLE. HsEg5 can be distinguished from NuMA by indirect immunofluorescence and Western blotting.

Adult↗