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Appelgren, Henrik
Publications (2 of 2) Show all publications
Appelgren, H., Kniola, B. & Ekwall, K. (2003). Distinct centromere domain structures with separate functions demonstrated in live fission yeast cells. Journal of Cell Science, 116(19), 4035-4042
Open this publication in new window or tab >>Distinct centromere domain structures with separate functions demonstrated in live fission yeast cells
2003 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 116, no 19, p. 4035-4042Article in journal (Refereed) Published
Abstract [en]

Fission yeast (Saccharomyces pombe) centromere DNA is organized in a central core region flanked on either side by a region of outer repeat (otr) sequences. The otr region is known to be heterochromatic and bound by the Swi6 protein whereas the central core region contains an unusual chromatin structure involving the histone H3 variant Cnp1 (S. pombe CENP-A). The central core is the base for formation of the kinetochore structure whereas the flanking region is important for sister centromere cohesion. We have previously shown that the ultrastructural domain structure of S. pombe centromeres in interphase is similar to that of human centromeres. Here we demonstrate that S. pombe centromeres are organized in cytologically distinct domains even in mitosis. Fluorescence in situ hybridization of fixed metaphase cells revealed that the otr regions of the centromere were still held together by cohesion even after the sister kinetochores had separated. In live cells, the central cores and kinetochores of sister chromosomes could be distinguished from one Another when they were subjected to mitotic tension. The function of the different centromeric domains was addressed. Transacting mutations affecting the kinetochore (nuf2) central core domain (mis6) and the heterochromatin domain (rik1) were analyzed in live cells. In interphase, both nuf2 and mis6 caused declustering of centromeres from the spindle pole body whereas centromere clustering was normal in rik1 despite an apparent decondensation defect. The declustering of centromeres in mis6 cells correlated with loss the Ndc80 kinetochore marker protein from the centromeres. Interestingly the declustered centromeres were still restricted to the nuclear periphery thus revealing a kinetochore-independent peripheral localization mechanism for heterochromatin. Time-lapse microscopy of live mis6 and nuf2-1 mutant cells in mitosis showed similar severe misaggregation phenotypes whereas the rik1 mutants showed a mild cohesion defect. Thus, S. pombe centromeres have two distinguishable domains even during mitosis, and our functional analyses support the previous observations that the kinetochore/central core and the heterochromatin domains have distinct functions both in interphase and mitosis.

National Category
Cell Biology
Identifiers
urn:nbn:se:sh:diva-15533 (URN)10.1242/jcs.00707 (DOI)000187395100022 ()12928332 (PubMedID)2-s2.0-0142042826 (Scopus ID)
Available from: 2012-02-21 Created: 2012-02-21 Last updated: 2017-12-07Bibliographically approved
Facanha, A. L., Appelgren, H., Tabish, M., Okorokov, L. & Ekwall, K. (2002). The endoplasmic reticulum cation P-type ATPase Cta4p is required for control of cell shape and microtubule dynamics. Journal of Cell Biology, 157(6), 1029-1039
Open this publication in new window or tab >>The endoplasmic reticulum cation P-type ATPase Cta4p is required for control of cell shape and microtubule dynamics
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2002 (English)In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 157, no 6, p. 1029-1039Article in journal (Refereed) Published
Abstract [en]

Here we describe the phenotypic characterization of the cta(4+) gene, encoding a novel member of the P4 family of P-type ATPases of fission yeast. The cta4Delta mutant is temperature sensitive and cold sensitive lethal and displays several morphological defects in cell polarity and cytokinesis. Microtubules are generally destabilized in cells lacking Cta4p. The microtubule length is decreased, and the number of microtubules per cell is increased. This is concomitant with an increase in the number of microtubule catastrophe events in the midzone of the cell. These defects are likely due to a general imbalance in cation homeostasis. Immunofluorescence microscopy and membrane fractionation experiments revealed that green fluorescent protein-tagged Cta4 localizes to the ER. Fluorescence resonance energy transfer experiments in living cells using the yellow cameleon indicator for Ca2+ indicated that Cta4p regulates the cellular Ca2+ concentration. Thus, our results reveal a link between cation homeostasis and the control of cell shape, microtubule dynamics, and cytokinesis, and appoint Ca2+ as a key ion in controlling these processes.

National Category
Cell Biology
Identifiers
urn:nbn:se:sh:diva-15802 (URN)10.1083/jcb.200111012 (DOI)000176188100013 ()12058018 (PubMedID)2-s2.0-0037054553 (Scopus ID)
Available from: 2012-03-08 Created: 2012-03-07 Last updated: 2017-07-20Bibliographically approved
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