EC Accepts, published online ahead of print on 14 August 2009

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Eukaryotic Cell doi:10.1128/EC.00062-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Cytoplasmic bulk flow propels nuclei in mature hyphae of Neurospora crassa

Silvia L. Ramos-García, Robert W. Roberson, Michael Freitag, Salomón Bartnicki-García, and Rosa R. Mouriño-Pérez^*

Departamento de Microbiología, División de Biología Experimental y Aplicada, Centro de Investigación Científica y Educación Superior de Ensenada, Ensenada, B. C. México; School of Life Sciences, Arizona State University, Tempe, Arizona, USA; Department of Biochemistry and Biophysics, Center for Genome Research and Biocomputing, Oregon State University, Corvallis, Oregon, USA

^* To whom correspondence should be addressed. Email: rmourino{at}cicese.mx.

In this work, we used confocal microscopy to evaluate nuclear dynamics in mature, growing hyphae of Neurospora crassa whose nuclei expressed histone H1-tagged GFP. In addition to the H1-GFP wild type strain (WT), we examined nuclear displacement (passive transport) in four mutants deficient in microtubule-related motor proteins (ro-1, ro-3, kin-1 and a ro-1 kin-1 double mutant). We also treated the WT strain with benomyl and cytochalasin A to disrupt microtubules and actin microfilaments, respectively. We found that the degree of nuclear displacement in the subapical regions of all strains correlated with hyphal elongation rate. The WT and the ro-1 kin-1 double mutant showed the highest correlation between nuclear movement and hyphal elongation. While most nuclei seemed to move forward passively presumably carried by cytoplasmic bulk flow, a small proportion of the movement detected was either retrograde or accelerated anterograde. The absence of a specific microtubule motor in the mutants ro-1, ro-3 or kin-1 did not prevent the anterograde and retrograde migration of nuclei; however in the ro-1 kin-1 double mutant retrograde migration was absent. In WT, almost all nuclei were elongated, while in all other strains a majority of nuclei were nearly spherical. With only one exception, a sizable exclusion zone was maintained between the apex and the leading nucleus. The ro-1 mutant showed the largest nucleus exclusion zone; only the treatment with cytochalasin A abolished the exclusion zone. In conclusion, the movement and distribution of nuclei in mature hyphae appear to be determined by a combination of forces, with cytoplasmic bulk flow being a major determinant. Probably, motor proteins play an active role in powering the retrograde or accelerated anterograde migrations of nuclei and may also contribute to passive anterograde displacement by binding nuclei to microtubules.