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Spindle positioning in human cells relies on proper centriole formation
and on the microcephaly proteins CPAP and STIL
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| Journal of Cell Science |
| Centrosome Biology Laboratory, Kitagawa Group |
Spindle positioning in human cells relies on proper centriole formation and on the microcephaly proteins CPAP and STIL
Daiju Kitagawa, Gregor Kohlmaier, Debora Keller, Petr Strnad, Fernando R. Balestra, Isabelle Flückiger and Pierre Gönczy
Journal of Cell Science, 124:3884-3893
DOI: 10.1242/jcs.089888 Featured in JCS "In This Issue" as 'Putting spindles in the right place'.
Patients with MCPH (autosomal recessive primary microcephaly) exhibit impaired brain development, presumably due to the compromised function of neuronal progenitors. Seven MCPH loci have been identified, including one that encodes CPAP (centrosome protein 4.1 associated protein). CPAP is a large coiled-coil protein enriched at the centrosome, which comprises two centrioles and surrounding pericentriolar material (PCM). CPAP depletion impairs centriole formation, whereas CPAP overexpression results in overly long centrioles. The mechanisms by which CPAP MCPH patient mutations affect brain development are not clear. Here, we identify CPAP protein domains critical for its centriolar localization, as well as for the elongation and the formation of centrioles. Furthermore, we demonstrate that conditions that resemble CPAP MCPH patient mutations compromise centriole formation in tissue culture cells. Using adhesive micropatterns, we reveal that such defects in centriole formation lead to a randomization of spindle position. Moreover, we demonstrate that the MCPH protein STIL (SCL/TAL1 interrupting locus) is also essential for centriole formation and for proper spindle position. Our findings are compatible with the notion that mutations in CPAP and STIL cause MCPH due to aberrant spindle positioning in progenitors cells during brain development.
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MCPH proteins are required for correct spindle positioning in human cells.
(A-B) Representative interphase (A) and mitotic (B) HeLa cells on a L-shape fibronectin micropattern (orange), stained with antibodies against Centrin-3 (red) and α-tubulin (green); DNA is shown in blue. Note that the cell spreads along the L-shaped micropatterned fibronectin substrate during interphase and orients along the hypothenus of the L-shape during mitosis.
(C) Schematic representation of mitotic spindle geometry on L-shape micropattern. The spindle is shown in green, centrosomes in red and chromosomes in blue. Spindle position was determined during metaphase-early anaphase as an angle as depicted, with 0° being defined as a parallel to the hypothenus of the L-shape.
(D-E) Synchronized HeLa cells either untreated (D), or treated with siRNAs against STIL (E) plated on fibronectin L-shape micropatterns (orange) and stained with antibodies against Centrin-3 (in red in the low magnification merge and in black and white in the ~2 fold magnified insets) and α-tubulin (green); DNA is shown in blue. The dashed lines indicate the position of mitotic spindles. Second right panels: schematic view of mitotic configurations. Right panels: frequency of angular distributions of spindle orientations in 15° increments, with the shading indicative of the frequency in each class.
(F) Speculative model suggesting how defective CPAP or STIL function reduces the pool of neuroepithelial progenitors in MCPH patients. Normally (left), progenitor cells predominantly divide symmetrically early during brain development, with the spindle being positioned parallel to the ventricular surface, thus maintaining the progenitor cell pool. When CPAP or STIL function is altered in MCPH patients (right), centriole formation is defective, which sometimes results in the spindle being positioned perpendicular to the ventricular surface, which would result in a reduction of the progenitor pool during early brain development.
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