Cytoplasmic Bulk Flow Propels Nuclei in Mature Hyphae of Neurospora crassa |
| |
Authors: | Silvia L Ramos-García Robert W Roberson Michael Freitag Salomón Bartnicki-García Rosa R Mouri?o-Pérez |
| |
Institution: | Departamento de Microbiología, División de Biología Experimental y Aplicada, Centro de Investigación Científica y Educación Superior de Ensenada, Ensenada, Mexico,1. School of Life Sciences, Arizona State University, Tempe, Arizona,2. Department of Biochemistry and Biophysics, Center for Genome Research and Biocomputing, Oregon State University, Corvallis, Oregon3. |
| |
Abstract: | We used confocal microscopy to evaluate nuclear dynamics in mature, growing hyphae of Neurospora crassa whose nuclei expressed histone H1-tagged green fluorescent protein (GFP). In addition to the H1-GFP wild-type (WT) strain, 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 strain and that the ro-1 kin-1 double mutant showed the highest correlation between nuclear movement and hyphal elongation. Although most nuclei seemed to move forward passively, presumably carried by the 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 the WT strain, almost all nuclei were elongated, whereas 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. Motor proteins probably 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.Organelle movement and positioning are important aspects of cell growth and differentiation (19, 20, 27, 35). Movement and positioning of nuclei are especially important because of their implications in mitotic divisions during hyphal growth and asexual sporulation (conidiation), as well as fertilization events leading to meiosis and ascospore formation during sexual development (1, 3, 33). In yeast, nuclei move comparatively short distances (20, 32), whereas in filamentous fungi nuclei are typically transported over long distances within hyphae (1, 34, 35).Movement of nuclei in fungal cells may be either an active or a passive process. Early studies of filamentous fungi showed nuclei uniformly distributed along the entire hypha; they appeared to move with the growing hyphal apex, keeping a more or less constant distance from the cell tip. Such evidence pointed to passive displacement of nuclei by cytoplasmic bulk flow (10-12, 24), a role confirmed in our recent study on the dynamics of the microtubular cytoskeleton (28) and supported by studies with injected lipid droplets (17). Upon the discovery of motor proteins and their role in nuclear migration and positioning in filamentous fungi, attention was primarily focused on the participation of motors in nuclear events, including the movement of nuclei during hyphal extension (15, 25, 26, 29, 37), while the role of cytoplasmic bulk flow was largely discounted or disregarded.Whereas much effort has been directed toward the characterization of the components involved in motor-driven nuclear transport, the relative importance of passive nuclear propulsion has remained an open question. For the purpose of distinguishing clearly between active migration and passive displacement, we will consider “migration” to mean an active, motor-dependent process, while “displacement” will refer to passive transport of nuclei within the hypha. “Movement” refers either to active or passive transport of nuclei through hyphae. Here, we used strains of Neurospora crassa whose nuclei were tagged with green fluorescent protein (GFP) to examine the dynamics and distribution of nuclei in growing hyphae. In addition to evaluating nuclear movement in a wild-type (WT) strain, we examined the dynamics of nuclear movement in mutants defective in microtubule-related motor proteins: a ro-1 mutant for its deficiency in the heavy chain of dynein, a ro-3 mutant deficient in the dynactin p150glued subunit, a kin-1 mutant deficient in conventional kinesin, and a ro-1 kin-1 a dynein-kinesin double mutant. We also tested the effect of drugs that inhibit specifically microtubules and actin microfilaments. Our study demonstrates that passive displacement plays a major role in nuclear dynamics in growing hyphae of N. crassa. |
| |
Keywords: | |
|
|