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Introduction: Anticentromere autoantibodies are associated with refractory IVF/ET failure, but causality is unclear. Experimental models are needed., Methods: Immature oocytes collected from 23-day-old mice were matured in vitro for 18 h in a culture medium containing an anti-human centromere protein A (CENP-A) polyclonal antibody, and those oocytes' maturity and chromosome/spindle structure were assessed., Results: Antibody exposure did not affect the germinal vesicle breakdown ratio but reduced the first polar body formation ratio by 13% at the highest concentration (70.0 µg/mL). Metaphase II (MII) oocytes were stained for chromosomes/spindles and grouped into aligned/barrel-like (AB), scattered/weakly-stained (SW), and condensed/absent (CA). Antibody exposure decreased AB and increased SW and CA in a dose-dependent manner. The AB/SW/CA percentages were 86/14/0, 86/14/0, 35/65/0, and 0/0/100 in the 0, 17.5, 35.0, and 70.0 µg/mL antibody groups, respectively (underlined values represent p < 0.05 compared with 0 µg/mL). Next, metaphase II oocytes were subjected to intracytoplasmic sperm injection, and the number of pronucleus/pronuclei (PN) was counted 6 h later. Antibody exposure decreased two pronuclei oocytes and increased non-two pronuclei oocytes dose-dependently. The percentages of 0/1/2/3 pronuclei oocytes were 43/0/57/0, 37/0/21/42, 16/28/48/8, and 91/4/4/0 in the 0, 17.5, 35.0, and 70.0 µg/mL groups, respectively., Conclusions: Anti-CENP-A antibody impaired a linear alignment of chromosomes at metaphase II and enhanced one or three PN formation after ICSI, which are similar to findings reported for infertile women with anticentromere autoantibodies., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: The clinical significance of common antinuclear antibody (ANA) patterns, such as nuclear homogenous and nuclear speckled patterns with their corresponding specific antibodies, has already been established. However, the clinical relevance of these uncommon ANA patterns have not been well elucidated and these patterns are therefore not reported by most clinical laboratories. We herein report some retrospective data analysis linking patients' clinical status to several uncommon ANA patterns., Methods: We retrieved and assessed the patient records for ANA reports generated in our hospital over a period of two years. All testing had been performed using the gold standard Indirect Immunofluorescence Assay., Results: Records of 1235 consecutive patients tested for ANA were reviewed. ANA was positive in 330 of these patients with 6.39% found to have uncommon nuclear, cytoplasmic or mitotic sub-patterns. The mitotic spindle (0.89%), cytoplasmic anti-mitochondrial antibodies (0.80%), followed by discrete nuclear dots-multiple (0.72%) were the dominating patterns, with a higher prevalence in females than in males. Systemic lupus erythematosus and rheumatoid arthritis were the two most common autoimmune disorders associated with mitotic spindle fibers and nuclear centromere and nuclear large/coarse speckled ANA patterns., Conclusion: The prevalence of these relatively uncommon ANA patterns was higher than expected. Further evaluation of these patterns along with their corresponding antibodies and their clinical utility must be encouraged. We trust this endeavour will provide diagnostic information in autoimmune and other disease conditions., (Copyright © 2021 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

BACKGROUND MAD2 is the gene controlling mitosis. Many studies have assessed MAD2 in various types of carcinoma. Antinuclear mitotic spindle apparatus antibody (MSA) and anticentromere antibody (ACA) are related mitotic antibodies, playing roles in autoimmune diseases and carcinomas, but the expression of MAD2, MSA, and ACA in SCLC is unclear. MATERIAL AND METHODS We enrolled 70 SCLC patients, 72 non-small cell lung cancer (NSCLC) patients, and 65 pulmonary nodule (PN) patients. MAD2 expression was measured through agarose electrophoresis and qt-PCR. Antinuclear mitotic spindle apparatus antibody (MSA) and anticentromere antibody (ACA) were detected by indirect immunofluorescence (IIF). RESULTS MAD2 was found both in SCLC and NSCLC. Interestingly, there was a significant difference found between SCLC and NSCLC using qt-PCR (P<0.05). The area under the ROC curve of MAD2 expression was 0.799, with medium diagnostic value. MAD2 expression was related to age, lymphatic metastasis, and survival time, but not with sex. The positivity for MSA and ACA by IIF assay were 37.20% and 34.00%, respectively, in the SCLC group, which were higher than in the NSCLC and pulmonary nodule groups (P<0.05). The kappa values of MSA and ACA with MAD2 expression were 0.73 and 0.65, respectively, with moderate consistency. Combining MAD2 with MSA and ACA enhanced the sensitivity and specificity for diagnosing SCLC. CONCLUSIONS MAD2 expression was found to be involved in carcinogenesis and prognosis of SCLC. The combination of MAD2 with MSA and ACA is useful for early diagnosis and shows promise in treatment of SCLC.

Antinuclear antibodies (ANA) are key biomarkers in the evaluation of rheumatic diseases. The prevalence and clinical significance of uncommon or rare patterns, particularly those directed at the mitotic spindle apparatus (MSA), are not well understood. We aimed to investigate the prevalence and clinical significance of anti-MSA patterns in a Colombian population.During 2013 and 2014, 113,491 consecutive determinations of ANA were studied for the presence of uncommon patterns. Clinical and laboratory data of anti-MSA positive patients were retrospectively collected and analyzed.Of the 113,491 patients tested, 60,501 (53%) were positive for ANA, of which 834 (1.3%) were positive for uncommon/rare patterns of ANA (anti-MSA in 592 cases). Of these 592 cases, complete data were available in 329 patients, of whom 116 had an established diagnosis. Anti-MSA antibodies were the only ANA positive test in 81% patients. At least one fine reactivity was identified in 19/116 (16.3%) of ANA-positive patients, of which anti-Ro was the most prevalent (18/116, 15.5%).The most frequent patterns were nuclear mitotic apparatus (NuMA) (56%) and MSA-2 (25%). The NuMA pattern had the highest ANA titers: mean 320 (range 80-2560) and behaved as monospecific antibodies. The most frequent systemic autoimmune diseases were Sjögren syndrome (SS) (18.1%), rheumatoid arthritis (RA) (13.8%), and systemic lupus erythematosus (SLE) (11%). Undifferentiated connective tissue disease (UCTD) was associated with the centrosome (P < .001), NuMA (P < .02) and MSA-2 (P < .45) patterns. Chronic idiopathic urticaria (CIU) was associated with the NuMA pattern (P < .02) and sensorineural hearing loss (SNHL) was associated with the MSA-2 (P < .001), centrosome (P < .68) and CENP-F (P < .38) patterns, previously unreported findings. Malignancies were found in 8 patients (50% were papillary thyroid cancer).In a large cohort of ANA determinations, uncommon patterns were found in around 1% of cases. The most frequent anti-MSA patterns found were NuMA and MSA-2. More than 50% of patients with anti-MSA had an associated CTD, mainly SS, RA and SLE, and anti-MSA behaved as monospecific antibodies. Other entities of presumed autoimmune origin, like CIU and SNHL, might be associated with these patterns.

Background: Mitotic spindle apparatus (MSA) antibodies are rare findings with undefined clinical significance in clinical research. We aimed at investigating the prevalence and clinical significance of anti-MSA antibodies in Chinese population., Methods: Between 2008 and 2013, a total of 180,180 patients were studied for the presence of anti-MSA antibodies. The clinical details and laboratory data of anti-MSA-positive patients were retrospectively collected and analyzed., Results: Of the 180,180 patients tested, 68,640 patients presented with positive antinuclear antibodies (ANAs, 38.10%), but only 32 patients with positive anti-MSA antibodies (0.018%). Diagnoses were established in 22 of 32 patients: 16 connective tissue diseases (CTDs), mainly Sjogren syndrome (SS, 5/16), rheumatoid arthritis (RA, 4/16), and systemic lupus erythematosus (SLE, 3/16), and 6 nonautoimmune conditions. The most frequent clinical symptoms of the anti-MSA-positive patients were arthralgia and eyes and mouth drying. Additionally, 70% of anti-MSA antibodies were not associated with other ANAs, however, when associated, the most frequent ANA was anti-SSA., Conclusions: Anti-MSA antibodies have a low prevalence and female gender predominance. Anti-MSA antibodies are primarily associated with CTDs, mainly SS, RA, and SLE. The presence of anti-MSA antibodies might be the unique serological markers of the CTDs, especially when anti-SSA, SSB, and dsDNA antibodies are negative, or the level of RF is low., (© 2016 Wiley Periodicals, Inc.)

Objective: Anti-NuMA1 and anti-NuMA2 antibodies are antinuclear antibodies (ANA) targeting the mitotic spindle apparatus. Our objective was to determine their clinical and immunological features and to review the literature available data., Patients and Methods: Between 2004 and 2008, 36,498 sera were analyzed for the presence of ANA, which included anti-NuMA1 and anti-NuMA2 antibodies. Clinical and immunological features of patients with positive anti-NuMA1 and anti-NuMA2 antibodies (titer> or =1/320) were retrospectively collected and analyzed. A review of the literature was secondly performed., Results: Out of the 36,498 sera analyzed, 10,585 sera were positive for ANA (29%). Out of ANA positive sera, 40 sera (0.38%) (40 different patients) were positive for anti-NuMA antibodies: 27 anti-NuMA1 (0.26%) and 13 anti-NuMA2 (0.12%). Compared to anti-NuMA2 positive patients, anti-NuMA1 positive patients were more often female (81.5% versus 46%; P=0.03), had more frequently a connective tissue disease (CTD) (40.7% versus 0%; P=0.016) and higher serum titers (877+/-466 versus 443+/-278; P=0.007). The anti-NuMA1 positive CTD were either Sjögren's syndrome (SS) (54.5%) or systemic lupus erythematosus (SLE) (45.5%). In the literature, 164 anti-NuMA positive patients (133 anti-NuMA1 and 31 anti-NuMA2) have been reported. Combining the reported cases to ours, up to 67.5% of anti-NuMA positive patients had an autoimmune disease, mostly pSS in 34% (31/90) and SLE in 31% (28/90). Anti-NuMA1 antibodies were the single positive ANA in 46% of anti-NuMA1 positive SS and 47% of anti-NuMA1 positive SLE, and anti-NuMA2 antibodies in 2/2 and 87.5%, respectively., Conclusion: Detection of anti-NuMA1 and anti-NuMA2 antibodies is very uncommon. When present, they are mostly associated with connective tissue disease, mainly Sjögren syndrome and systemic lupus. Clinicians may be aware that in these latter conditions, anti-NuMA antibodies may be the single serological marker., (2010 Elsevier B.V. All rights reserved.)

Objective: To evaluate the effects of tumor necrosis factor-alpha (TNF-alpha) on porcine oocyte maturation, spindle dynamics, and chromosome alignment., Design: Controlled, prospective study., Setting: University hospital and IVF research laboratory., Animal(s): Ovaries collected from slaughtered prepubertal gilts., Main Outcome Measure(s): Oocyte maturation rate and cytoskeleton distribution. MATERIALS AND METHOD(S): Immature porcine oocytes (GV) were exposed to TNF-alpha at a concentration of 0 (as a control), 1, 5, 10, 100, 200, or 600 ng/mL in M199 medium. Oocytes were cultured for 24 hours to the pre-MI stage or 44 hours to the MII stage. After in vitro maturation for 44 hours, the rates of GV oocytes reaching MII stage were assessed, and MII oocytes were fixed for further examination of the cytoskeleton and the chromosomal distribution., Result(s): The TNF-alpha concentration at 5 ng/mL decreased the porcine oocyte maturation rate compared with the control after culture for 44 hours, whereas exposure to 10 or 100 ng/mL TNF-alpha resulted in a significant increase in the frequency of defective spindles or abnormal microfilament distribution. Exposed to 200 ng/mL, TNF-alpha caused a significantly higher abnormality rate of chromosome alignment when compared with the controls., Conclusion(s): Exposure of porcine oocytes to an elevated TNF-alpha concentration clearly caused a reduction in their maturation from GV stage to MII stage and increased the proportion of oocytes with abnormal chromosome alignment and cytoskeleton structure., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Antibodies against the nuclear mitotic spindle apparatus protein (NuMA) are infrequently detected during antinuclear antibodies testing on HEp-2 cells. In a series of 428 psoriatic patients anti-NuMA antibodies were found only in a patient, at a titer of 1:640, without any apparent clinical relevance. The significance of anti-NuMA is not yet known and is briefly reviewed, also in consideration of potential therapeutic implications. Although biologic drugs targeting tumor necrosis factor-alpha have been associated with the development of non-organ specific autoantibodies and rare reports of autoimmune phenomena, infliximab was well tolerated in this patient and caused no changes in autoantibody titers.

The most important mitotic apparatus (MA) antigens are centrosome (CE), nuclear mitotic apparatus (NuMA-1, NuMA-2), midbody, and centromere F (CENP-F). We studied associations of anti-MA antibodies with other autoantibodies and their clinical significance. A total of 6270 patients were studied for the presence of anti-MA antibodies on HEp-2 cells. Sera positive for anti-MA were tested for anti-extractable nuclear antigens (ENA) antibodies. Anti-MA antibodies were detected in 56 (45 females and 11 males) of 6270 sera (0.9%). Of these 56, NuMA-1 was found in 23, NuMA-2 in 7, CE in 20, CENP-F in 5, and CENP-F/centrosome in 1 case. Anti-NuMA-1 were associated with anti-ENA antibodies (p < 0.001). Diagnoses were established in 43/56 patients: 22 connective tissue diseases, 7 infections, 6 autoimmune hepatitis, 3 vasculitis, 3 primary antiphospholipid syndrome, 1 malignancy, and 1 fever of unknown origin. The differential diagnosis of anti-NuMA-1-positive patients must include Sjögren's syndrome, while patients with anti-CE antibodies must be observed for HCV infection.

The Int-6 protein has been originally identified as the product of a mouse gene being a frequent integration site of the mouse mammary tumour virus. Here, we show that reducing Int-6 expression by RNA interference in HeLa cells markedly alters mitosis progression. Defects in spindle formation, chromosome segregation and cytokinesis were observed. These abnormalities of mitosis completion are correlated with an inhibition of cyclin B-Cdk1 kinase activity, due to a prolonged inhibitory phosphorylated state of Cdk1. In line with this observation, the Wee1 tyrosine kinase that negatively controls Cdk1 was less efficiently inactivated during G2 in Int-6-depleted cells. These findings support the notion that the oncogenic properties associated with alteration of Int-6 originate from chromosomal instability.

Centrosome is the major microtubule organizing center in mammalian cells that plays a critical role in a variety of cellular events by the microtubule arrays emanating from it. Despite its significance, the molecular mechanisms underlying the structure and function of the centrosome are still not clear. Herein we describe the identification of three isotypes of human ninein by expression library screening with autoimmune sera from CREST patients. All three ninein isotypes exhibit centrosomal localization throughout the cell cycle when GFP-tagged fusion proteins are expressed transiently in mammalian cells. Construction of serial deletions of GFP-tagged ninein reveals that a stretch of three leucine zippers with a flanking sequence is required and sufficient for centrosomal targeting. Overexpression of ninein results in mislocalization of gamma-tubulin, recruiting it to ectopic (noncentrosomal) ninein-containing sites which are not active in nucleating microtubules. In these cells, nucleation of microtubules from the centrosome is also inhibited. These results thus suggest a regulatory role for ninein in microtubule nucleation.

Objective: To determine the clinical significance of anti-NuMA and anti-HsEg5 antibodies in a group of patients affected with rheumatic diseases., Materials and Methods: Indirect immunofluorescence on HEp-2000 cells at serum dilution of 1:40 was used to examine 26 sera which had previously showed a "mitotic spindle" fluoroscopic pattern type during laboratory routine., Results: 21 sera (80,7%) were identified with NuMA and 5 (19,3%) with HsEg5 patterns alone or associated with other ANA patterns. However only patients with isolated positivity and that is 15 with NuMA and 4 with HsEg5 stainings were included in this study. Of the NuMA positive patients 5 were affected with arthropathies associated to different forms of thyroiditis, 2 with seronegative arthritis, 2 with antiphospholipid syndrome, 1 with systemic lupus erythematosus (SLE), 1 with rheumatoid arthritis, 1 with sicca syndrome, 1 with undifferentiated connective tissue disease, 1 with Mycoplasma pneumoniae infection and 1 with retinal thrombosis. Of the HsEg5 positive patients 3 were affected with SLE and 1 with seronegative arthritis., Conclusions: NuMA does not prevail in any defined rheumatic disease, while HsEg5 staining were more frequent (75%) in patients affected with SLE all of whom showing high antibody titres.

Recently, the large filamentous striated-muscle protein titin has been observed in non-muscle cells, and, in one instance, has been proposed to have a nuclear function as a chromosomal component contributing to structure and elasticity. In this study, we sought to further characterize the presumptive nuclear isoform of titin. Immunofluorescence microscopy with multiple titin-specific monoclonal antibodies shows localization to the nucleus in interphase cells and to the spindle machinery in mitotic cells in all cell types examined; localization to condensed chromosomes is not observed. An abundant 700-kDa phosphoprotein is the predominant species immunoprecipitated with these antibodies. Sequencing of peptide fragments of the immunopurified protein reveals identity to AHNAK, a nuclear phosphoprotein, an identification that was confirmed by Western blot analysis with antibodies to AHNAK and peptide fragmentation patterns. Sequence comparison suggests similarities between the repetitive heptad phi+/-phiP+/-phi+/- motif in AHNAK and the PEVK region of titin, potentially explaining the cross-reactivity observed between AHNAK antibodies and titin antibodies. Interestingly, although some AHNAK antibodies stain interphase nuclei, no evidence of mitotic spindle localization is seen, suggesting that the identity of the protein at the latter location is more closely related to titin than AHNAK. This concept is further supported by observations that cell lines not expressing AHNAK have similar antititin antibody localization to the mitotic spindle. We conclude that (1) multiple titin antibodies, particularly those recognizing the PEVK region, cross-react with AHNAK, and (2) the mitotic spindle staining observed with antititin antibodies is most likely due to the association of titin or a titin-like molecule with this structure., (Copyright 2001 Wiley-Liss, Inc.)

Background: Human autoantibodies to proteins of the mitotic apparatus have demonstrated clinical utility and usefulness as molecular probes for identification and characterization of novel autoantigens, as exemplified by autoantibodies to centromere proteins. In contrast, there have been very few reports of autoantibodies with reactivity to antigens located along mitotic chromosome arms, but not in interphase nuclei. The purpose of this study was to identify and characterize autoantibodies with reactivity to mitotic chromosomal antigens (MCAs) located exclusively on mitotic chromosome arms, and to determine if patients with these autoantibodies have common clinical features., Methods: Routine immunofluorescence screening of serum samples referred for antinuclear antibody investigation over a 10-year period was used to identify autoantibodies to MCAs. MCAs were identified by exclusive immunofluorescence staining of mitotic chromosome arms with no staining of interphase nuclei. MCA-reactive sera were further characterized for patterns of staining on mitotic chromosome arms and sensitivities to chemical and enzymatic treatments, and for one of these sera, its ability to abrogate progression through mitosis when microinjected into cells., Results: Of 60,000 sera screened for antinuclear antibodies by immunofluorescence, we identified three IgG autoantibodies reacting exclusively to MCAs. The anti-MCA autoantibodies did not react with condensed chromatin in spermatozoa or in apoptotic HeLa cells. Reactivity of all three sera was abrogated by treatment with protease, but not RNase, indicating that the MCAs are protein in nature and do not contain RNA epitopes. The three anti-MCA antibodies seem to react to three different antigens because they gave different patterns of staining of chromosome arms, reacted with chromosomes in different stages of mitosis, and displayed different sensitivities to treatment with DNase 1, salt, and phosphatases. Phosphatase treatment suggests that MCA1 and MCA2 contain serine/threonine phosphoepitope(s) and MCA3 tyrosine phosphoepitope(s). Loss of MCA2 reactivity to DNase 1 treatment and its retention after salt extraction suggests that it is a chromosomal scaffold protein. Sensitivity of all three MCAs to acid suggests that they are histone-like or histone-associated proteins., Conclusions: We report the identification of three novel MCA-reactive sera. Patient diagnoses included discoid lupus erythematosus, chronic lymphocytic leukemia, Sjögren's syndrome, and polymyalgia rheumatica. The reactivity of anti-MCA antibodies with phosphoepitopes is likely to explain restriction of immunofluorescence staining to chromosome arms during mitosis. Microinjection of MCA1-reactive antibodies led to metaphase arrest, without any change in morphology of the mitotic spindle or metaphase chromosomes suggesting that MCA1 may have a role in sister chromatid separation.

Background: The mitotic spindle apparatus (MSA) is a unique structure of microtubules and associated proteins involved in the segregation and reorganisation of chromosomes during cell division. Autoantibodies to the MSA (anti-MSA) are reported to occur rarely, but are easily identified during the immunofluorescent detection of anti-nuclear antibodies (ANA), and are generally reported as part of that investigation., Aims: As the clinical significance of these antibodies is unknown, our aim was to identify the clinical features of subjects identified with anti-MSA, and in a subset investigate the co-association with organ specific anti-thyroid antibodies., Methods: All ANA results from the three major immunology laboratories serving South Australia between January 1993 and June 1998 were retrospectively reviewed to identify anti-MSA subjects. Clinical details were extracted from hospital or general practice records using a standard proforma. Thyroid autoantibodies were measured using standard technique. A control group of consecutive ANA positive, anti-MSA negative individuals had anti-thyroid antibodies measured. Statistical comparison used chi2 test., Results: Fifty-five subjects (43F) were identified with mean age 59.8 (range 17-91); 39 had specific diagnoses, with 16 identified as part of non-specific investigations. 'Arthritis' broadly accounted for the largest group, transient inflammatory arthritis n=7, degenerative joint disease n=6, rheumatoid arthritis n=5. Adenocarcinoma and mesothelioma accounted for one case each. Thirty-two subjects had anti-thyroid antibodies tested, with ten of 21 and two of 11 positive among the groups with anti-MSA titre >1:80 and <1:40 respectively, chi2=2.7, p=0.1. Anti-thyroid antibodies were detected more frequently among the high titre anti-MSA group (ten of 21) compared with high titre positive ANA, negative anti-MSA group (two of 11), RRisk 4.4, chi2=5.34, p=0.02., Conclusion: This study confirmed the relative rarity of anti-MSA and that its association is primarily with rheumatic diseases. The coincidence of mesothelioma is novel with only two previous reports of malignancy and anti-MSA. The co-association of high titre anti-MSA and thyroid autoantibodies suggest that the latter should be a follow up investigation if the former is identified as part of an investigative screen.

Background: Serum antibodies in scleroderma patients are generally directed against the nucleolus and centromeres. A small proportion of patients have serum antibodies to the centrioles and mitotic apparatus., Objective: To determine the prevalence of serum autoantibodies against the mitotic apparatus in scleroderma patients., Material and Methods: Sera from 113 patients with various forms of scleroderma were tested for antinuclear antibodies by indirect immunofluorescence on HEp-2 cells. The specificity of the antibodies was determined by Western blot., Results: Only two scleroderma sera recognized the mitotic apparatus. Western blot results showed that in both cases the target was an about 235 kDa protein corresponding to the NuMA determinant. Affinity-purified anti-NuMa antibodies were used to perform immunolocalization in synchronized HEp-2 cells using scanning laser confocal microscopy. The anti-NuMA autoantibodies recognized the mitotic asters but neither the centrioles nor the microtubules., Conclusion: Our data suggest that anti-NuMA autoantibodies may be devoid of clinical significance in scleroderma. However, they remain useful as probes in cell biology studies.

Genetic evidence has shown the presence of a common spindle pole organiser in Physarum amoebae and plasmodia. But the typical centrosome and mitosis observed in amoebae are replaced in plasmodia by an intranuclear mitosis devoid of any structurally defined organelle. The fate of gamma-tubulin and of another component (TPH17) of the centrosome of Physarum amoebae was investigated in the nuclei of synchronous plasmodia. These two amoebal centrosomal elements were present in the nuclear compartment during the entire cell cycle and exhibited similar relocalisation from metaphase to telophase. Three preparation methods showed that gamma-tubulin containing material was dispersed in the nucleoplasm during interphase. It constituted an intranuclear thread-like structure. In contrast, the TPH17 epitope exhibited a localisation close to the nucleolus. In late G2-phase, the gamma-tubulin containing elements condensed in a single organelle which further divided. Intranuclear microtubules appeared before the condensation of the gamma-tubulin material and treatment with microtubule poisons suggested that microtubules were required in this process. The TPH17 epitope relocalised in the intranuclear spindle later than the gamma-tubulin containing material suggesting a maturation process of the mitotic poles. The decondensation of the gamma-tubulin material and of the material containing the TPH17 epitope occurred immediately after telophase. Hence in the absence of a structurally defined centrosome homologue, the microtubule nucleating material undergoes a cycle of condensation and decondensation during the cell cycle.

In tumour cell lines, the resistance of cancer cells to a variety of structurally unrelated chemotherapeutic drugs is termed multidrug-resistance or MDR. We reported previously [6] that MDR leukemic cells displayed nuclear texture changes, as assessed by image cytometry. The nature of these changes remained uncertain but they could be associated with alterations of the nuclear matrix which could serve an important role in DNA organization and chromatin structure. Therefore, we have compared the textural features observed in G0/G1 nuclei from human leukemic CEM cells and their MDR variant CEM-VLB, after staining of either DNA by Feulgen method or nuclear matrix by immunodetection of NuMA antigen on DNase treated samples. Chromatin or NuMA distributions within the nucleus were evaluated by image cytometry. Changes in textural parameters indicate that modifications of NuMA distribution observed in MDR cells are parallel to those observed at the whole chromatin level (i.e., a more decondensed and coarse texture with increase of Energy and Long-run sections and decrease of Contrast and Short-run sections). Moreover, Optical Densities measurements indicate that MDR cells seem to contain less NuMA, a datum confirmed by immunoblotting of nuclear proteins. In conclusion, chromatin changes observed by image cytometry in drug-resistant human leukemic CEM cells appear associated with modifications of the nuclear matrix structure.

The Xenopus centrosome protein kinase pEg2, involved in spindle assembly, binds to microtubules polymerized in vitro. We have developed a method to investigate the affinity of purified recombinant pEg2 protein for the cellular mitotic spindle. Briefly, cells grown on coverslips are fixed, permeabilized, and incubated with recombinant pEg2 protein. Localization of the protein is revealed by probing with a specific monoclonal antibody that recognizes recombinant but not endogenous pEg2. Using this method we show that recombinant pEg2 binds to microtubules in vitro, while, in vivo, pEg2 localized only to the mitotic spindle and not the interphase microtubule network. We also demonstrate that the catalytic activity of pEg2 is not necessary for its binding ability. This technique can be used to analyze the binding of various tagged proteins to cellular mitotic spindle., (Copyright 1998 Academic Press.)

The monoclonal antibody 78H6 recognises an 85 kDa component of the yeast spindle pole body. Here we identify and characterise this component as Spc72p, the product of YAL047C. The sequence of SPC72 contains potential coiled-coil domains; its overexpression induced formation of large polymers that were strictly localised at the outer plaque and at the bridge of the spindle pole body. Immunoelectron microscopy confirmed that Spc72p was a component of these polymers. SPC72 was found to be non-essential for cell growth, but its deletion resulted in abnormal spindle positioning, aberrant nuclear migration and defective mating capability. Precisely, deletion of SPC72 resulted in a decreased number of astral microtubules: early in the cell cycle only few were detectable, and these were unattached to the spindle pole body in small-budded cells. Later in the cell cycle few, if any, remained, and they were unable to align the spindle properly. We conclude that Spc72p is not absolutely required for nucleation per se, but is needed for normal abundance and stability of astral microtubules.

Autoantibodies directed to a variety of cellular antigens and organelles are a feature of autoimmune diseases. They have proven useful in a clinical setting to establish diagnosis, estimate prognosis, follow disease progression, alter therapy, and initiate new investigations. Cellular and molecular biologists have used autoantibodies as probes to identify molecules involved in key cellular processes. One of the most interesting sets of autoantibodies are those that target antigens within the mitotic apparatus (MA). The MA includes chromosomes, spindle microtubules and centrosomes. The identification, localization, function, and clinical relevance of MA autoantigens is the focus of this review.

Using RNA in situ hybridization to reveal cytoplasmic localization patterns of mRNAs in cultured cells, we noted unexpected staining of a cytoplasmic component in telophase cells. Control experiments revealed that the anti-digoxin-specific antibody was responsible for this staining. Because the staining was observed only at a position where both daughter cells are still connected, we identified the stained component as the midbody. This was confirmed by double staining of cells with anti-digoxin and anti-alpha-tubulin antibodies. We concluded that anti-digoxin-specific antibody shows crossreactivity with a component present in the midbody.

Objective: To characterize human autoantigen-antibody systems related to the mitotic poles and spindles., Methods: Thirty-seven human sera with autoantibodies staining mitotic poles and spindles in indirect immunofluorescence (IIF) studies were further characterized by immunofluorescence on mitotic cells and by immunoblotting and immunoprecipitation. Clinical diagnoses meeting the American College of Rheumatology criteria were based on chart review and interview with the corresponding physicians., Results: Two autoantibody systems reactive with mitotic poles and spindles were defined. Type 1 nuclear mitotic apparatus (NuMA-1) antibodies were identified in the serum of 30 patients. Interphase cells showed a fine, speckled, nuclear staining, while mitotic cells had bright staining of the rim of the centrosomes and light staining of the spindles proximal to the centrosomes. In telophase, the staining shifted from the centrosomes to the reforming nuclei. On immunoblotting, anti-NuMA-1 sera reacted with a 210-kd protein. The reactivity of these sera was identified (with the aid of reference antibodies) as the previously described NuMA antigen-antibody system. Clinical information was available for only 17 of the 30 patients with anti-NuMA-1; of these, 17 (53%) had clinical and lip biopsy findings that met the criteria for Sjögren's syndrome. NuMA-2 antibodies were found in the sera of 7 patients. Interphase cells showed no nuclear or cytoplasmic staining, but mitotic cells had brightly stained poles and spindles. At anaphase/telophase, staining shifted to the midbody and the intercellular bridge. Anti-NuMA-2 sera immunoprecipitated a protein of 116 kd. This group of patients was more heterogeneous and had both systemic and organ-specific autoimmune diseases., Conclusions: NuMA protein (here called NuMA-1) and a 116-kd protein (here called NuMA-2) are the major targets of the autoimmune response in the mitotic apparatus, since most of the selected sera (based on IIF staining of the mitotic spindles and poles) recognized 1 of these 2 antigens.

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.

We have demonstrated that dynamic redistribution of nuclear-mitotic apparatus (NuMA) protein in the cell cycle is correlated temporally and spatially with its biochemical modifications. In interphase, NuMA behaves solely as a 220 kDa nuclear matrix-associated protein. After initiation of DNA condensation during mitosis, NuMA is phosphorylated by Cdc2 kinase into a 240 kDa form which is transported quickly to the centrosomal region. Once cells have passed the metaphase-anaphase transition, the 240 kDa form of NuMA either becomes a 180 kDa truncated form which is fated to be degraded completely before mitotic exit, or returns to the 220 kDa form that relocates to the daughter nuclei and remains throughout interphase. Apparently, a proteolytic enzyme is activated during the late stages of mitosis. After induction of a 180 kDa form of NuMA in interphase HeLa cells by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, nuclear apoptotic phenomena including chromatin condensation, DNA fragmentation, and micronucleation were observed. However, the same treatment did not induce apoptosis in mitotic phase-arrested HeLa cells. The 180 kDa form of NuMA was demonstrated to be a truncated product, at least lacking the tail domain. When HL60 cells were stimulated by diverse apoptosis inducers such as camptothecin, staurosporine, cycloheximide, and A23187, the extent of NuMA cleavage to produce a 180 kDa product was comparable with the degree of oligonucleosomal laddering. NuMA cleavage is likely to be a consequence of the onset of apoptosis. The intact 220 kDa NuMA functions in interphase cells to retain the nuclear structural integrity. Additionally, NuMA appears to act as a nuclear structural target for a death protease during apoptosis.

Cell division was arrested by injection of a preimmune rabbit serum, B-61, into PtK(2) cells during interphase and prometaphase. Identical results were obtained by injection of whole B-61 antiserum and of antibodies affinity-purified from the serum against a 66-kDa PtK(2) cell polypeptide. When injected into interphase cells, the antibodies arrested further development and cell division. When injected into prometaphase and metaphase cells, spindles shortened and poles moved together at a rate of 0.2-0.4 mu m/min, approximately half the rate of anaphase A chromosome movements in normally dividing PtK(2) cells. Chromosomes decondensed and cells did not reenter division. Both whole antisera and affinity-purified antibodies stained antigens diffusely localized throughout the cytoplasm in dividing and interphase cells. These results suggest that the 66-kDa antigen is a nonspindle protein that may regulate mitotic progression in PtK(2) cells.

NuMA, a 238 kDa protein present in the nucleus during interphase, translocates to the spindle poles in mitosis. NuMA plays an essential role in mitosis, since microinjection of the NuMA SPN-3 monoclonal antibody causes mitotic arrest and micronuclei formation. We have mapped the approximate position of the epitopes of six monoclonal NuMA antibodies using recombinant NuMA fragments. The SPN-3 epitope has been located to residues 255-267 at the C-terminus of the first helical subdomain of the central rod domain and several residues crucial for antibody binding have been identified. To gain insight into the ultrastructure of NuMA, several defined fragments, as well as the full-length recombinant protein, were expressed in Escherichia coli and purified to homogeneity. They were then characterized by chemical cross-linking, circular dichroism spectra and electron microscopy. The results directly reveal the tripartate structure of NuMA. A long central rod domain is flanked by globular end domains. The rod is 207 nm long and is at least 90% alpha-helical. It reflects a double-stranded coiled-coil with the alpha-helices arranged parallel and in register. The NuMA protein thus forms the longest coiled-coil currently known. Our analyses reveal no indication that recombinant NuMA assembles into filaments or other higher order structures.

Centrosomes are critical for the nucleation and organization of the microtubule cytoskeleton during both interphase and cell division. Using antibodies raised against sea urchin sperm flagellar microtubule proteins, we characterize here the presence and behavior of certain components associated with centrosomes of the surf clam Spisula solidissima and cultured mammalian cells. A Sarkosyl detergent-resistant fraction of axonemal microtubules was isolated from sea urchin sperm flagella and used to produce monoclonal antibodies, 16 of which were specific- or cross-specific for the major polypeptides associated with this microtubule fraction: tektins A, B and C, acetylated alpha-tubulin, and 77 and 83 kDa polypeptides. By 2-D isoelectric focussing/SDS polyacrylamide gel electrophoresis the tektins separate into several polypeptide spots. Identical spots were recognized by monoclonal and polyclonal antibodies against a given tektin, indicating that the different polypeptide spots are isoforms or modified versions of the same protein. Four independently derived monoclonal anti-tektins were found to stain centrosomes of S. solidissima oocytes and CHO and HeLa cells, by immunofluorescence microscopy. In particular, the centrosome staining of one monoclonal antibody specific for tektin B (tekB3) was cell-cycle-dependent for CHO cells, i.e. staining was observed only from early prometaphase until late anaphase. By immuno-electron microscopy tekB3 specifically labeled material surrounding the centrosome, whereas a polyclonal anti-tektin B recognized centrioles as well as the centrosomal material throughout the cell cycle. Finally, by immunoblot analysis tekB3 stained polypeptides of 48-50 kDa in isolated spindles and centrosomes from CHO cells.

Serum samples from 49 children with acute Mycoplasma pneumoniae infection were screened for the presence of antibodies to mitotic spindle apparatus. None of these serum samples showed such antibodies at a screening dilution of 1:40, though anticentriolar antibodies at titres of 1:320 were observed in two children with acute cerebellar dysfunction. Anticentriolar antibodies may play a part in the pathogenesis of CNS disease associated with M pneumoniae infection.

We have employed human autoantibodies to characterize a novel cell cycle-regulated nuclear protein, provisionally designated p330d (doublet polypeptide of 330 kDa). The expression and intracellular distribution of this protein was followed throughout the cell cycle using immunofluorescence microscopy, laser confocal microscopy, immunoelectron microscopy and flow cytometry. p330d was expressed only in proliferating cells and began accumulating in the nucleus during early S phase. The protein reached maximum expression levels during G2/M. In situ extractions with detergent, salt and nucleases failed to abolish the nuclear staining of interphase cells, suggesting a tight binding of p330d to the nuclear matrix during interphase. p330d was concentrated in the kinetochores during prophase but was relocated to the spindle midzone at the onset of anaphase. By late telophase, it was localized predominantly in the intercellular bridge regions flanking the midbody and disappeared gradually as the daughter cells separated. Immunoblotting analysis showed that the autoimmune sera recognized a doublet of 330 kDa, and affinity-purified antibodies from this doublet reproduced the fluorescence staining pattern of the whole serum. We propose that p330d is a novel member of the class of 'chromosomal passenger' proteins, which are associated transiently with centromeres during early mitosis and are then redistributed to other sites of the mitotic apparatus after the metaphase/anaphase transition. Possible in vivo functions for p330d and related proteins might include roles in centromere/kinetochore maturation and assembly, chromosome segregation, central spindle stabilization and cytokinesis.

The distribution of antigens to two antibodies (Bx63 and Rb188) that associate to Drosophila melanogaster centrosomes has been investigated in the nematode Parascaris. By western blot analysis both antibodies identify in Parascaris polypeptides of the same molecular mass as in Drosophila (Rb188 a 185 kDa antigen and Bx63 185 kDa and 66 kDa antigens). By immunocytochemistry we show that the centrosomes of Parascaris contain the 185 kDa antigen recognized by polyclonal Rb188 and monoclonal Bx63 antibodies. In addition, Bx63 reveals cytoplasmic midzone structures, not found in Drosophila, that display a cell cycle-dependent organization in embryos. These structures, which most probably contain the 66 kDa antigen revealed by Bx63, appear at the onset of anaphase as fibrillar-like structures that during anaphase form a ring-like structure encircling the equatorial plane of the blastomere. Before furrowing, the antigen participates in the formation of the midbody and associates with convergent polar microtubules. After blastomere division, Bx63 signal persists as a single body between the daughter cells. The analysis of chilled and nocodazole-treated embryos suggests that the localization of the midzone Bx63 antigen is dependent on non-kinetochore microtubules. Inhibition of furrowing by cytochalasin B shows that the antigen persists after the disassembly of microfilaments. Cytological observations of contractile ring and Bx63 ring assembly indicate that both structures do not simultaneously colocalize at the equatorial zone. The data suggest a spindle-dependent distribution of the Bx63 antigen during cytokinesis. We discuss the participation of this antigen in the organization of the midbody before furrowing, and consider the possible relevance of the midbody with respect to cell to cell communication during early development in nematodes.

A phosphorylated epitope is differentially expressed at the kinetochores of chromosomes in mitotic cells and may be involved in regulating chromosome movement and cell cycle progression. During prophase and early prometaphase, the phosphoepitope is expressed equally among all the kinetochores. In mid-prometaphase, some chromosomes show strong labeling on both kinetochores; others exhibit weak or no labeling; while in other chromosomes, one kinetochore is intensely labeled while its sister kinetochore is unlabeled. Chromosomes moving toward the metaphase plate express the phosphoepitope strongly on the leading kinetochore but weakly on the trailing kinetochore. This is the first demonstration of a biochemical difference between the two kinetochores of a single chromosome. During metaphase and anaphase, the kinetochores are unlabeled. At metaphase, a single misaligned chromosome can inhibit further progression into anaphase. Misaligned chromosomes express the phosphoepitope strongly on both kinetochores, even when all the other chromosomes of a cell are assembled at the metaphase plate and lack expression. This phosphoepitope may be involved in regulating chromosome movement to the metaphase plate during prometaphase and may be part of a cell cycle checkpoint by which the onset of anaphase is inhibited until complete metaphase alignment is achieved.

Circulating autoantibodies against the nuclear mitotic apparatus (NuMA) protein have been detected in sera of patients with a variety of connective tissue diseases. A close relation of anti-NuMA antibodies to define subsets of these diseases could not be established. This is the first description of a patient with vitiligo, autoimmune thyroiditis and a high titer of antibodies against NuMA on Hep-2 cells. The possibility of a beginning of multiple autoimmune syndrome cannot be excluded.

We have identified a novel M(r) 36,000 protein (MSA-36) that has a complex cell cycle dependent distribution. This protein is first detected in interphase nuclei just prior to the onset of chromosome condensation. MSA-36 is found along condensing chromosomes and is a component of the centromere through metaphase. At anaphase, this protein is no longer detected in association with the chromosomes but appears at the forming stembodies and subsequently within the intercellular bridge at either side of the midbody. At the completion of cell division, the amount of MSA-36 in the bridge appears to decline concurrent with the appearance of this protein briefly within the reforming nucleus. To investigate whether MSA-36 is an active component of the chromosome or a passive passenger protein, we studied the behaviour of this protein in cells exhibiting premature chromosome condensation and in cells during and following recovery from mitotic arrest. These studies suggest that MSA-36 is not essential for a variety of major chromosome-associated events.

We have used monoclonal antibodies raised against isolated native calf thymus centrosomes to probe the structure and composition of the pericentriolar material. To distinguish prospective antibodies as specific to conserved elements of this material, we screened clones by their identification of microtubule organizing centers (MTOCs) in different animal and plant cells. Among the clonal antibodies that reacted with MTOCs in both plant and mammalian cells, we describe one (mAb 6C6) that was found to immunostain centrosomes in a variety of bovine and human cells. In cycling cells this signal persisted through the entire cell cycle. Microscopy showed that the mAb 6C6 antigen was a component of the pericentriolar material and this was confirmed by biochemical analysis of centrosomes. Using immunoblot analysis of protein fractions derived from purified components of centrosomes, we have characterized the mAb 6C6 antigen as a 180 kDa polypeptide. We conclude that we have identified a protein component permanently associated with the pericentriolar material. Surprisingly, monoclonal antibody 6C6 also stained other mitotic organelles in mammalian cells, in a cell-cycle-dependent manner. During prometaphase and metaphase the antibody stained both centrosomes and kinetochores. At the onset of anaphase the kinetochore-specific staining dissociated from chromosomes and was subsequently redistributed onto a newly characterized organelle, the telophase disc while the centrosomal stain remained intact. It is not known if the 180 kDa centrosomal protein itself redistributes during mitosis, or if the pattern observed represents other antigens with shared epitopes. The pericentriolar material is thought to be composed of conserved elements, which appeared very early during the evolution of eukaryotes. Our results strongly suggest that mAb 6C6 identifies one of these elements.

Tektins were originally described as a set of three filamentous proteins (tektin A, B and C) associated with the walls of axonemal microtubules of sea urchin sperm. Using affinity-purified polyclonal antibodies raised against tektins of two sea urchin species, Lytechinus pictus and Strongylocentrotus purpuratus, we looked for tektin-like components in microtubule systems other than axonemes. By immunofluorescence microscopy we observed labeling of meiotic spindles in eggs of the surf clam Spisula solidissima and in several mammalian cell lines. In Spisula eggs the tektin-like antigens were still associated with the spindles after about 95% of the tubulin had been removed via a calcium/cold treatment. In pig kidney epithelial cells the tektin-like antigen appeared to be associated with bundles of calcium-stable spindle microtubules. By SDS-PAGE immunoblot the affinity-purified anti-tektins recognized several polypeptides in tubulin-depleted spindle remnants of Spisula eggs: A approximately 52 kDa, 1 M KCl-resistant component was identified by the antibody raised against tektin C from S. purpuratus, a approximately 48 kDa component was recognized by the antibody specific for tektin A from L. pictus, and three polypeptide bands (approximately 64 kDa, approximately 100 kDa and greater than 200 kDa) were detected by the antibody specific for tektin C from L. pictus. Only the latter antibody, however, stained Spisula spindles by immunofluorescence microscopy. We further report that the sensitivity of antibody recognition of proteins on immunoblots is dependent on the purity of sodium dodecyl sulfate.

To identify kinesin-related proteins that may be important for mitotic function in embryonic and tissue culture cells we have generated polyclonal antibodies to two synthetic peptides corresponding to conserved regions of the kinesin motor domain. In Xenopus eggs we have identified a family of microtubule-binding proteins, recognized by one or both affinity-purified peptide antibodies but not by monoclonal antibodies that recognize conventional kinesin heavy chain. Like kinesin, most of these proteins bind to microtubules only upon addition of AMP-PNP or nucleotide depletion and are released upon subsequent addition of ATP. At least one protein, however, exhibits markedly distinct properties, binding readily to microtubules in the absence of AMP-PNP and/or nucleotide depletion. We also report that, unlike antibodies to conventional kinesin, the peptide antibodies to the kinesin motor domain immunofluorescently label spindles and kinetochores in mitotic tissue culture cells, suggesting that kinesin-like proteins may have important roles in chromosome movement and mitosis.

Using the monoclonal antibody MA-01, which recognizes a 210-kDa protein in cell-free extracts, spindle and cytoplasmic microtubules were visualized in budding yeast, Saccharomyces cerevisiae. In additional, a spot-like staining was found beneath the plasma membrane, revealing in part correlation with F-actin distribution. This pattern was common for cells of all cell-cycle stages. The interaction of the protein recognized by MA-01 with microtubules was confirmed in the double labeling with a polyclonal antitubulin antibody and by the sensitivity of intranuclear structures stained by MA-01 to the microtubule disrupting drug nocodazole.

The molecular composition of two morphologically distinct microtubule-organizing centers (MTOCs) was compared by probing with monoclonal antibodies raised against (i) nucleus-associated bodies (NABs) isolated in a complex with nuclei from the cellular slime mold Dictyostelium discoideum and (ii) mammalian mitotic spindles isolated from Chinese hamster ovary (CHO) cells. The staining patterns observed by immunofluorescence microscopy in whole CHO cells and Dictyostelium amoebae showed that the distribution of thirteen MTOC antigens is heterogeneous. Not all antibodies recognized the MTOC in both interphase and mitosis. Most of the anti-MTOC antibodies cross-reacted with other cellular organelles such as nuclei, Golgi apparatus-like aggregates and cytoskeletal elements. Two antibodies, CHO3 and AX3, recognized phosphorylated epitopes present in both mammalian centrosomes and Dictyostelium NABs. On immunoblots, most of the antibodies showed multiple bands, often of high molecular weight, indicating that the antigenic determinants are shared among different molecules. One antibody inhibited the regrowth of microtubules onto centrosomes in vitro after addition of exogenous tubulin to detergent-lysed CHO cells on coverslips; this antibody binds to an antigen(s) that might be essential for the microtubule-nucleating activity of centrosomes. These observations demonstrate that molecular components in different MTOCs exhibit a variety of distinct subcellular localizations and functional properties, and that some antigenic molecules have been conserved among morphologically distinct MTOCs.

Autoantibody to mitotic spindle associated antigen (MSA) was examined in sera from patients with positive mycoplasma pneumoniae particle agglutination (PA) test along with various autoimmune diseases and healthy subjects by means of indirect immunofluorescence (IF) using HEp-2 cells as substrates. Anti-MSA antibody was identified in 9 patients sera including 7 out of 180 sera with positive mycoplasma pneumoniae PA test, one with systemic lupus erythematosus (SLE) and one with scleroderma (PSS) respectively. Five of 9 sera with positive anti-MSA antibody contained anti-nuclear antibody (ANA) including 4 with speckled staining ANA and one with homogeneous + nucleolar staining ANA. Two collagen diseases sera positive for anti-MSA antibody showed negative mycoplasma pneumoniae PA test. In addition, there was no correlation between the presence of anti-MSA antibody and the titers of mycoplasma pneumoniae PA test. Our results indicated that anti-MSA antibody was not limited to such connective tissue diseases as previously described by other workers. It would be speculated that mycoplasma pneumoniae infection might induce anti-MSA antibody production.

As cells enter mitosis, centrosomes undergo many transformations and become associated with different molecules in a stage-specific manner. We have developed a protocol for immunofluorescence staining with four antibody probes that can help us to follow the interaction of centrosomal components during mitosis. The cells were first stained with a human autoimmune serum (5051); a monoclonal anti-phosphocentrosomal antibody (CHO3); and an antitubulin antibody. Localization of the antibodies was detected using rhodamine-, fluorescein- and AMCA-conjugated second antibodies, respectively. After photographing marked mitotic cells, coverslips were soaked with 0.2 M glycine-HCl at pH 1.0 for 1 h to release all antibodies bound to the structures. The same cells were re-stained with a human autoantibody (SP-H) specific for spindle poles and a fluorescein-conjugated second antibody. This allowed us to compare the subcellular distribution of three kinds of centrosomal antigens in a single cell. Mitotic PtK1 cells treated with either nocodazole or taxol included microtubule-containing cytoplasmic foci and parallel bundles of short microtubules at the cell periphery. All the centrosomal antibodies stained the same one or two dots corresponding to structures labeled by the tubulin antibody. CHO3 also revealed extra cytoplasmic foci, whereas the SP-H antigen was additionally localized at one end of the free microtubule bundles. As the microtubules reorganized into bipolar spindles during the recovery from drug treatment, the CHO3 and SP-H antigens coalesced into the spindle poles where the 5051 antigen was located, suggesting that centrosomal antigens become associated with spindle poles through very different recruitment pathways.