Nuclear defects in hyperelongated cells at high temperature conditions. The light field image on the left shows a hyperelongated cell. The image on the right is a DAPI staining of the same hyperelongated cell complex. The arrowhead indicates the location of the nucleus. Arrows indicate the cell complexes that lack a nucleus.

C. neoformans actin expression at 37°C in KN99α and KN99α rom2. F-actin is identified with rhodamine-phalloidin. Actin is distributed throughout the wild type cell. White arrows indicate actin localization at the cell periphery and at the bud tips of KN99α rom2 hyperelongated cells. The black arrow indicates the region of the cell that is defective for cytokinesis indicated by a lack of cell wall formation between the cell complexes.

Sensitivity to nocodazole was assessed using KN99α, KN99α rom2, and KN99α rom2+ROM2. Cultures were grown overnight and serial dilutions were plated on YPD with 2 µM nocodazole. Plates were grown for 2 days at 25°C, 30°C or 37°C to detect growth inhibition.

Micrographs and diagrams of C. neoformans morphology after 24 hours of growth are presented for KN99α and KN99α rom2. A. KN99α cells grown at 30°C and 37°C. Scale bars represent 20 µM. B. The KN99α cells appear to have a round morphology. The left image is a micrograph and the right image is a diagram. C. KN99α rom2 cells grown at 30°C and 37°C cluster together. At 37°C growth conditions, the hyperelongated morphology is also observed. White arrows indicate cells that are “tear” shaped and form a cluster. The black arrow indicated a hyperelongated complex. 100× magnification. D. KN99α rom2 cells appear as a cluster of 3–4 cells per complexes. The left image is a micrograph and the right image is a diagram. E. KN99α rom2 cells also demonstrate a hyperelongated phenotype at 37°C that forms a chain of cell complexes. The arrowhead indicates the region of the hyperelongated cell defective for cytokinesis, indicated by the lack of cell wall formation between to cell complexes. The left image is a micrograph and the right image is a diagram of the cell morphology.

Individual cell localization of actin and microtubules. Actin staining with rhodamine-phalloidin of both KN99α and KN99α rom2 cells at 30°C and 37°C. Microtubules were identified using a TAT1 antibody and staining with goat anti mouse secondary antibody as described in the materials and methods.

Microtubule localization at 37°C conditions. Microtubules are distributed throughout wild type cells. Microtubules are found in localized areas of KN99α rom2 hyperelongated cells. The distribution of cellular material between mother and daughter cell is well defined for wild type cells but is enlarged over a greater area in hyperelongated cells. Arrowheads indicate the location of microtubule patches. Microtubules are distributed throughout mother and daughter cells in KN99α. However, microtubules are localized to the neck region and periphery of the hyperelongated KN99α rom2 cells. Arrows indicate microtubule extensions between mother and daughter cells.