Desmosomal Antigens Are Not Recognized by the Majority of Pemphigus Autoimmune Sera

Sera from 7 patient with pemphigus vulgaris and both mouse and rabbit antisera against bovine epidermal desmosomes contained antibodies that bound to cell surface components of the spinous layer of bovine epidermis. The antidesmosomal sera show significant binding to purified desmosomal proteins in an enzyme-linked immunosorbent assay (ELISA). Two of 7 pemphigus sera bound to desmosomal protein-coated microtiter plates at low dilution titers. Two of 6 normal human sera also bound to desmosomal protein-coated microtiter plates at titers comparable to those of the pemphigus sera. Indirect immunofluorescent labeling of frozen sections of monkey esophagus revealed striking differences in the distribution of pemphigus antigens and desmosomal constituents. Pemphigus antisera produced rather uniform fluorescence around the borders of spinous cells of the esophageal epithelium, while anti-desmosomal antibodies bound in a punctate pattern. Anti-desmosomal antibodies labeled cells of the basal layer in a strongly punctate pattern. Only 1 pemphigus serum appreciably labeled basal cells. Two of 3 anti-desmosomal antisera bound avidly in the upper differentiating layers of the epithelium. Pemphigus antibodies did not. Pemphigus sera that reacted with desmosomal proteins in ELISA were absorbed by affinity chromatography on immobilized desmosomal proteins. This treatment did not alter the immunofluorescent labeling patterns produced by these sera. From these results we conclude that the pemphigus autoantibodies studied here bind to epithelial cell surface antigens which are distinguishable from the structural components of desmosomes

Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells

Cdc20 is a substrate adaptor and activator of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase whose activity is required for anaphase onset and exit from mitosis. A green fluorescent protein derivative, Cdc20–GFP, bound to centrosomes throughout the cell cycle and to kinetochores from late prophase to late telophase. We mapped distinct domains of Cdc20 that are required for association with kinetochores and centrosomes. FRAP measurements revealed extremely rapid dynamics at the kinetochores (t1/2 = 5.1 s) and spindle poles (t1/2 = 4.7 s). This rapid turnover is independent of microtubules. Rapid transit of Cdc20 through kinetochores may ensure that spindle checkpoint signaling at unattached/relaxed kinetochores can continuously inhibit APC/CCdc20 targeting of anaphase inhibitors (securins) throughout the cell until all the chromosomes are properly attached to the mitotic spindle

Elevating the level of Cdc34/Ubc3 ubiquitin-conjugating enzyme in mitosis inhibits association of CENP-E with kinetochores and blocks the metaphase alignment of chromosomes

Cdc34/Ubc3 is a ubiquitin-conjugating enzyme that functions in targeting proteins for proteasome-mediated degradation at the G1 to S cell cycle transition. Elevation of Cdc34 protein levels by microinjection of bacterially expressed Cdc34 into mammalian cells at prophase inhibited chromosome congression to the metaphase plate with many chromosomes remaining near the spindle poles. Chromosome condensation and nuclear envelope breakdown occurred normally, and chromosomes showed oscillatory movements along mitotic spindle microtubules. Most injected cells arrested in a prometaphase-like state. Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure. The elevation of Cdc34 protein levels in early mitosis selectively blocked centromere protein E (CENP-E), a mitotic kinesin, from associating with kinetochores. Other proteins, including two CENP-E–associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores. Proteasome inhibitors did not affect the prometaphase arrest induced by Cdc34 injection. These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation

Merotelic Kinetochore Orientation Is a Major Mechanism of Aneuploidy in Mitotic Mammalian Tissue Cells

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 μM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2–5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout

Haspin inhibitors reveal centromeric functions of Aurora B in chromosome segregation

Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), providing a docking site for the Aurora B complex at centromeres. Aurora B functions to correct improper kinetochore–microtubule attachments and alert the spindle checkpoint to the presence of misaligned chromosomes. We show that Haspin inhibitors decreased H3T3ph, resulting in loss of centromeric Aurora B and reduced phosphorylation of centromere and kinetochore Aurora B substrates. Consequently, metaphase chromosome alignment and spindle checkpoint signaling were compromised. These effects were phenocopied by microinjection of anti-H3T3ph antibodies. Retargeting Aurora B to centromeres partially restored checkpoint signaling and Aurora B–dependent phosphorylation at centromeres and kinetochores, bypassing the need for Haspin activity. Haspin inhibitors did not obviously affect phosphorylation of histone H3 at serine-10 (H3S10ph) by Aurora B on chromosome arms but, in Aurora B reactivation assays, recovery of H3S10ph was delayed. Haspin inhibitors did not block Aurora B localization to the spindle midzone in anaphase or Aurora B function in cytokinesis. Thus, Haspin inhibitors reveal centromeric roles of Aurora B in chromosome movement and spindle checkpoint signaling

Rapid exchange of mammalian topoisomerase IIα at kinetochores and chromosome arms in mitosis

Astable cell line (GT2-LPk) derived from LLC-Pk was created in which endogenous DNA topoisomerase IIα (topoIIα) protein was downregulated and replaced by the expression of topoIIα fused with enhanced green fluorescent protein (EGFP–topoIIα). The EGFP–topoIIα faithfully mimicked the distribution of the endogenous protein in both interphase and mitosis. In early stages of mitosis, EGFP–topoIIα accumulated at kinetochores and in axial lines extending along the chromosome arms. During anaphase, EGFP–topoIIα diminished at kinetochores and increased in the cytoplasm with a portion accumulating into large circular foci that were mobile and appeared to fuse with the reforming nuclei. These cytoplasmic foci appearing at anaphase were coincident with precursor organelles of the reforming nucleolus called nucleolus-derived foci (NDF). Photobleaching of EGFP–topoIIα associated with kinetochores and chromosome arms showed that the majority of the protein rapidly exchanges (t1/2 of 16 s). Catalytic activity of topoIIα was essential for rapid dynamics, as ICRF-187, an inhibitor of topoIIα, blocked recovery after photobleaching. Although some topoIIα may be stably associated with chromosomes, these studies indicate that the majority undergoes rapid dynamic exchange. Rapid mobility of topoIIα in chromosomes may be essential to resolve strain imparted during chromosome condensation and segregation

Developing immortal cell lines from Xenopus embryos, four novel cell lines derived from Xenopus tropicalis

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Gorbsky, G. J., Daum, J. R., Sapkota, H., Summala, K., Yoshida, H., Georgescu, C., Wren, J. D., Peshkin, L., & Horb, M. E. Developing immortal cell lines from Xenopus embryos, four novel cell lines derived from Xenopus tropicalis. Open Biology, 12(7), (2022): 220089, https://doi.org/10.1098/rsob.220089.The diploid anuran Xenopus tropicalis has emerged as a key research model in cell and developmental biology. To enhance the usefulness of this species, we developed methods for generating immortal cell lines from Nigerian strain (NXR_1018, RRID:SCR_013731) X. tropicalis embryos. We generated 14 cell lines that were propagated for several months. We selected four morphologically distinct lines, XTN-6, XTN-8, XTN-10 and XTN-12 for further characterization. Karyotype analysis revealed that three of the lines, XTN-8, XTN-10 and XTN-12 were primarily diploid. XTN-6 cultures showed a consistent mixed population of diploid cells, cells with chromosome 8 trisomy, and cells containing a tetraploid content of chromosomes. The lines were propagated using conventional culture methods as adherent cultures at 30°C in a simple, diluted L-15 medium containing fetal bovine serum without use of a high CO2 incubator. Transcriptome analysis indicated that the four lines were distinct lineages. These methods will be useful in the generation of cell lines from normal and mutant strains of X. tropicalis as well as other species of Xenopus.This work was supported by Whitman fellowships to G.J.G. from the Marine Biological Laboratory, by grant no. 1645105 to G.J.G. and MEH from the National Science Foundation and by grant no. P40OD010997 from the Office of the Director, National Institutes of Health. L.P. has been supported by grant no. R01HD073104 from the National Institute of Child Health and Development