A Fungal Kinesin Required for Organelle Motility, Hyphal Growth, and Morphogenesis

Abstract

A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformationmediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkö rper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkö rper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubulebased motor protein is essential for normal positioning of the Spitzenkö rper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.

Dynein and dynactin deficiencies affect the formation and function of the Spitzenkörper and distort hyphal morphogenesis of Neurospora crassa

“…Since mutations in kinesin are known to affect Spk formation (Seiler et al, 1997 ;Wu et al, 1998), it was proposed that cytoplasmic microtubules are oriented with their plus ends towards the apex (Lehmler et al, 1997 ;Seiler et al, 1997Seiler et al, , 1999. By the same reasoning, our studies showing that deficiency in cytoplasmic dynein or dynactin affects Spk formation could lead us to the opposite conclusion, namely that the microtubules are oriented with their minus ends towards the apex.…”

“…Our present observations with the dynein-deficient ropy mutants lend support to the notion that the microtubule cytoskeleton plays a major role in the formation and behaviour of the Spk. Similarly, Wu et al (1998) showed that kinesin was essential for normal positioning of the Spk in hyphae of N. haematococca. All this leads us to conclude that microtubule-associated motor proteins are necessary for maintenance of a high growth rate, a rather steady Spk and a near-perfect hyphoid shape.…”

“…Cytoplasmic dyneins and members of the kinesin superfamily are the main motor enzymes involved in vesicle translocation along microtubules (Haimo & Thaler, 1994 ;Hirokawa, 1998). There is now growing evidence that both kinesins and cytoplasmic dyneins are involved in the traffic of secretory vesicles in fungal hyphae (Seiler et al, 1997 ;Wu et al, 1998 ;Inoue et al, 1998). Cytoplasmic dyneins are multisubunit enzymes involved in transport of membranous organelles towards the minus end of microtubules (Paschal et al, 1987 ;Schroer & Sheetz, 1991 ;Hirokawa, 1998).…”

Universal and Unique Features of Kinesin Motors: Insights from a Comparison of Fungal and Animal Conventional Kinesins

“…While all animal kinesin knock-outs that have been performed so far (C. elegans, D. melanogaster, mouse) were lethal because of damage to neural tissues (Gho et al, 1992;Saxton et al, 1991;Patel et al, 1993;Tanaka et al, 1998) all three fungal kinesin null mutants from N. crassa (Seiler et al, 1997), N. haematococca (Wu et al, 1998) and Ustilago maydis (Lehmler et al, 1997) are viable and display informative phenotypes. In N. crassa the most prominent feature of the kinesin null mutant is its reduced hyphal growth speed (1.4 vs. 6.8 cm/day, Seiler et al, 1997).…”

“…While both of these were lethal, some less severe mutations in the Drosophila KHC gene were viable (Hurd and Saxton, 1996). In contrast, conventional kinesin is nonessential in fungi (Seiler et al, 1997;Wu et al, 1998;Lehmler et al, 1997), and its deletion yields informative phenotypes.…”