Cytoplasmic dynein is a microtubule-associated motor protein with several putative subcellular functions. Sequencing of the gene (DHC1) for cytoplasmic dynein heavy chain of the filamentous ascomycete, Nectria haematococca, revealed a 4,349-codon open reading frame (interrupted by two introns) with four highly conserved P-loop motifs, typical of cytoplasmic dynein heavy chains. The predicted amino acid sequence is 78.0% identical to the cytoplasmic dynein heavy chain of Neurospora crassa, 70.2% identical to that of Aspergillus nidulans and 24.8% identical to that of Saccharomyces cerevisiae. The genomic copy of DHC1 in N. haematococca wild-type strain T213 was disrupted by inserting a selectable marker into the central motor domain. Mutants grew at 33% of the wild-type rate, forming dense compact colonies composed of spiral and highly branched hyphae. Major cytological phenotypes included (1) absence of aster-like arrays of cytoplasmic microtubules focused at the spindle pole bodies of post-mitotic and interphase nuclei, (2) limited post-mitotic nuclear migration, (3) lack of spindle pole body motility at interphase, (4) failure of spindle pole bodies to anchor interphase nuclei, (5) nonuniform distribution of interphase nuclei and (6) small or ephemeral Spitzenkorper at the apices of hyphal tip cells. Microtubule distribution in the apical region of tip cells of the mutant was essentially normal. The nonuniform distribution of nuclei in hyphae resulted primarily from a lack of both post-mitotic nuclear migration and anchoring of interphase nuclei by the spindle pole bodies. The results support the hypothesis that DHC1 is required for the motility and functions of spindle pole bodies, normal secretory vesicle transport to the hyphal apex and normal hyphal tip cell morphogenesis.

REFERENCES

Aist
J. R.
(
1995
).
Independent nuclear motility and hyphal tip growth.
Can. J. Bot
73
,
122
–.
Aist
J. R.
,
Bayles
C. J.
(
1991
).
Ultrastructural basis of mitosis in the fungus Nectriahaematococca (sexual stage of Fusariumsolani) I.
Asters. Protoplasma
161
,
111
122
Aist
J. R.
,
Bayles
C. J.
,
Tao
W.
,
Berns
M. W.
(
1991
).
Direct experimental evidence for the existence, structural basis and function of astral forces during anaphase B in vivo.
J. Cell Sci
100
,
279
288
Aist
J. R.
,
Berns
M. W.
(
1981
).
Mechanics of chromosome separation during mitosis in Fusarium (fungi imperfecti): New evidence from ultrastructural and laser microbeam experiments.
J. Cell Biol
91
,
446
458
Allan
V.
(
1995
).
Membrane traffic motors.
FEBS Lett
269
,
101
106
Bartnicki-Garcia
S.
,
Hergert
F.
,
Gierz
G.
(
1989
).
Computer simulation of fungal morphogenesis and the mathematical basis for hyphal (tip) growth.
Protoplasma
153
,
46
57
Barton
N. R.
,
Goldstein
L. S. B.
(
1996
).
Going mobile: Microtubule motors and chromosome segregation.
Proc. Nat. Acad. Sci. USA
93
,
1735
1742
Beckwith
S. M.
,
Morris
N. R.
(
1995
).
Cytoplasmic dynein is missing at the tip in nudG, a nuclear migration mutant in Aspergillus nidulans.
Mol. Biol. Cell
6
,
5
–.
Berns
M. W.
,
Aist
J. R.
,
Wright
W. H.
,
Liang
H.
(
1992
).
Optical trapping in animal and fungal cells using a tunable, near-infrared titanium-sapphire laser.
Exp. Cell Res
198
,
375
378
Berns
M. W.
,
Wright
W. H.
,
Steubing
R. W.
(
1991
).
Laser microbeam as a tool in cell biology.
Int. Rev. Cytol
129
,
1
44
Bruno
K. S.
,
Tinsley
J. H.
,
Minkle
P. F.
,
Plamann
M.
(
1996
).
Genetic interactions among cytoplasmic dynein, dynactin, and nuclear distribution mutants of Neurosporacrassa.
Proc. Nat. Acad. Sci. USA
93
,
4775
4780
Carminati
J. L.
,
Stearns
T.
(
1997
).
Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex.
J. Cell Biol
138
,
629
641
Dunne
P. W.
,
Oakley
B. R.
(
1988
).
Mitotic gene conversion, reciprocal recombination and gene replacement at the benA, beta-tubulin, locus of Aspergillusnidulans.
Mol. Gen. Genet
213
,
339
345
Eshel
D.
(
1995
).
Functional dissection of the dynein motor domain.
Cell Motil. Cytoskel
32
,
133
135
Eshel
D. L.
,
Urrestarazu
L. A.
,
Vissers
S.
,
Jauniaux
J.-C.
,
van Vliet-Reedijk
J. C.
,
Planta
R. J.
,
Gibbons
I. R.
(
1993
).
Cytoplasmic dynein is required for normal nuclear segregation in yeast.
Proc. Nat. Acad. Sci. USA
90
,
11172
11176
Gaglio
T.
,
Saredi
A.
,
Bingham
J. B.
,
Hasbani
M. J.
,
Gill
S. R.
,
Schroer
T. A.
,
Compton
D. A.
(
1996
).
Opposing motor activities are required for the organization of the mammalian mitotic spindle pole.
J. Cell Biol
132
,
399
414
Gaglio
T.
,
Dionne
M. A.
,
Compton
D. A.
(
1997
).
Mitotic spindle poles are organized by structural and motor proteins in addition to centrosomes.
J. Cell Biol
138
,
1055
1066
Gepner
J.
,
Li
M.-G.
,
Ludmann
S.
,
Kortas
C.
,
Boylan
K.
,
Iyadurai
S. J. P.
,
McGrail
M.
,
Hays
T. S.
(
1996
).
Cytoplasmic dynein function is essential in Drosophilamelanogaster.
Genetics
142
,
865
878
Gibbons
I. R.
(
1995
).
Dynein family of motor proteins: Present status and future questions.
Cell Motil. Cytoskel
32
,
136
144
Goodson
H. V.
,
Valetti
C.
,
Kreis
T.
(
1997
).
Motors and membrane traffic.
Curr. Opin. Cell Biol
9
,
18
28
Hagan
I.
,
Yanagida
M.
(
1997
).
Evidence for cell cycle-specific, spindle pole body-mediated, nuclear positioning in the fission yeast Schizosaccharomyces pombe.
J. Cell Sci
110
,
1851
1866
Heald
R.
,
Tournebize
R.
,
Blank
T.
,
Sandaltzopoulos
R.
,
Becker
P.
,
Hyman
A.
,
Karsenti
E.
(
1996
).
Self-organization of microtubules into biopolar spindles around artificial chromosomes in Xenopus egg extracts.
Nature
382
,
420
425
Hyman
A. A.
(
1989
).
Centrosome movement in the early divisions of Caenorhabditis elegans: a cortical site determining centrosome position.
J. Cell Biol
109
,
1185
1193
Holzbaur
E. L. F.
,
Vallee
R. B.
(
1994
).
Dynein: Molecular structure and cellular function.
Annu. Rev. Cell Biol
10
,
339
372
Howard
R. J.
,
Aist
J. R.
(
1977
).
Effects of MBC on hyphal tip organization, growth, and mitosis of Fusariumacuminatum, and their antagonism byD2O.
Protoplasma
92
,
195
210
Inoue
S.
,
Aist
J. R.
,
Yoder
O. C.
,
Turgeon
B. G.
(
1996
).
Cytoplasmicdynein heavy chain is required for normal morphology and growth rate of the filamentous fungus Nectria haematococca.
Mol. Biol. Cell
7
,
402
–.
Jensen
C. G.
,
Aist
J. R.
,
Bayles
C. J.
,
Bollard
S. M.
,
Jensen
L. C. W.
(
1991
).
Ultrastructural basis of mitosis in the fungus Nectria haematococca (sexual stage of Fusariumsolani) III. Intermicrotubule bridges.
Protoplasma
161
,
137
149
Keller
N. P.
,
Bergstrom
G. C.
,
Yoder
O. C.
(
1991
).
Mitotic stability of transforming DNA is determined by its chromosomal configuration in the fungus Cochliobolusheterostrophus.
Curr. Genet
19
,
227
233
Kellogg
D. R.
,
Moritz
M.
,
Alberts
B. M.
(
1994
).
The centrosome and cellular organization.
Ann. Rev. Biochem
63
,
639
674
Koonce
M. P.
,
Samso
M.
(
1996
).
Overexpression of cytoplasmic dynein's globular head causes a collapse of the interphase microtubule network in Dictyostelium.
Mol. Biol. Cell
7
,
935
948
Koonce
M. P.
(
1996
).
Making a connection: the ‘other’ microtubule end.
Cell Motil. Cytoskel
35
,
85
93
Li
M.-G.
,
McGrail
M.
,
Serr
M.
,
Hays
T. S.
(
1994
).
Drosophila cytoplasmic dynein, a microtubule motor that is asymmetrically localized in the oocyte.
J. Cell Biol
126
,
1475
1494
Li
Y.-Y.
,
Yeh
E.
,
Hays
T.
,
Bloom
K.
(
1993
).
Disruption of mitotic spindle orientation in a yeast dynein mutant.
Proc. Nat. Acad. Sci. USA
90
,
10096
10100
Merdes
A.
,
Cleveland
D. W.
(
1997
).
Pathways of spindle pole formation: Different mechanisms; conserved components.
J. Cell Biol
138
,
953
956
Merdes
A.
,
Ramyar
K.
,
Vechio
J. D.
,
Cleveland
D. W.
(
1996
).
A complex of NUMA and cytoplasmic dynein is essential for mitotic spindle assembly.
Cell
87
,
447
458
Miao
V. P. W.
,
Mathews
D. E.
,
VanEtten
H. D.
(
1991
).
Identification and chromosomal locations of a family of cytochrome P-450 genes for pisatin detoxification in the fungus Nectriahaematococca.
Mol. Gen. Genet
226
,
214
223
Norrander
J.
,
Kempe
T.
,
Messing
J.
(
1983
).
Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis.
Gene
26
,
101
106
Paschal
B. M.
,
Vallee
R. B.
(
1987
).
Retrograde transport by the microtubule-associated protein MAP 1C.
Nature
330
,
181
183
Petes
T. D.
,
Hill
C. W.
(
1988
).
Recombination between repeated genes in microorganisms.
Annu. Rev. Genet
22
,
147
168
Pfarr
C. M.
,
Coue
M.
,
Grissom
P. M.
,
Hays
T. S.
,
Porter
M. E.
,
McIntosh
J. R.
(
1990
).
Cytoplasmic dynein is localized to kinetochores during mitosis.
Nature
345
,
263
265
Plamann
M.
,
Minke
P. F.
,
Tinsley
J. H.
,
Bruno
K. S.
(
1994
).
Cytoplasmic dynein and actin-related protein Arp1 are required for normal nuclear distribution in filamentous fungi.
J. Cell Biol
127
,
139
149
Raudaskoski
M.
,
Mao
W.-Z.
,
Yli-Mattila
T.
(
1994
).
Microtubule cytoskeleton in hyphal growth. Response to nocodazole in a sensitive and a tolelant strain of the homobasidiomycete Schizophyllumcommune.
Eur. J. Cell Biol
64
,
131
141
Rodionov
V. I.
,
Borisy
G. G.
(
1997
).
Self-centering activity of cytoplasm.
Nature
386
,
170
173
Rupes
I.
,
Mao
W.-Z.
,
Åström
H.
,
Raudaskoski
M.
(
1995
).
Effects of nocodazole and brefeldin A on microtubule cytoskeleton and membrane organization in the homobasidiomycete Schizophyllumcommune.
Protoplasma
185
,
212
221
Schnapp
B. J.
,
Reese
T. S.
(
1989
).
Dynein is the motor for retrograde axonal transport of organelles.
Proc. Nat. Acad. Sci. USA
86
,
1548
1552
Schroeder
C. C.
,
Fok
A. K.
,
Allen
R. D.
(
1990
).
Vesicle transport along microtubular ribbons and isolation of cytoplasmic dynein from Paramecium.
J. Cell Biol
111
,
2553
2562
Schroer
T. A.
(
1994
).
Structure function and regulation of cytoplasmic dynein.
Curr. Opin. Cell Biol
6
,
69
73
Seiler
S.
,
Nargang
F. E.
,
Steinberg
G.
,
Schliwa
M.
(
1997
).
Kinesin is essential for cell morphogenesis and polarized cell secretion in Neurospora crassa.
EMBO J
26
,
3025
3034
Shaw
S. L.
,
Yeh
E.
,
Maddox
P.
,
Salmon
E. D.
,
Bloom
K.
(
1997
).
Astral microtubule dynamics in yeast: A microtubule-based searching mechanism for spindle orientation and nuclear migration into the bud.
J. Cell Biol
139
,
985
994
Shortle
D.
,
Novick
P.
,
Botstein
D.
(
1984
).
Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene.
Proc. Nat. Acad. Sci. USA
81
,
4889
4893
Steele
G. C.
,
Trinci
A. P. J.
(
1977
).
Effect of temperature and temperature shifts on growth and branching of a wild type and temperature sensitive colonial mutant (Cot 1) of Neurosporacrassa.
Arch. Microbiol
113
,
43
48
Steuer
E. R.
,
Wordeman
L.
,
Schroer
T. A.
,
Sheetz
M. P.
(
1990
).
Localization of cytoplasmic dynein to mitotic spindles and kinetochores.
Nature
345
,
266
268
Taga
M.
,
Murata
M.
(
1994
).
Visualization of mitotic chromosomes in filamentous fungi by fluorescence staining and fluorescence in situ hybridization.
Chromosoma
103
,
408
413
That
T. C. C.-T.
,
Rossier
C.
,
Barja
F.
,
Turian
G.
,
Roos
U.-P.
(
1988
).
Induction of multiple germ tubes in Neurosporacrassa by antitubulin agents.
Eur. J. Cell Biol
46
,
68
79
Tinsley
J. H.
,
Minke
P. F.
,
Bruno
K. S.
,
Plamann
M.
(
1996
).
p150Glued, the largest subunit of the dyneinactin complex, is nonessential in Neurospora but required for nuclear distribution.
Mol. Biol. Cell
7
,
731
742
Upshall
A.
(
1986
).
Genetic and molecular characterization of arg B+transformants of Aspergillus nidulans.
Curr. Genet
10
,
593
599
Vaisberg
E. A.
,
Grissom
P. M.
,
McIntosh
J. R.
(
1996
).
Mammalian cells express three distinct dynein heavy chains that are localized to different cytoplasmic organelles.
J. Cell Biol
133
,
831
842
Vaisberg
E. A.
,
Koonce
M. P.
,
McIntosh
J. R.
(
1993
).
Cytoplasmic dynein plays a role in mammalian mitotic spindle formation.
J. Cell Biol
123
,
849
858
Vallee
R. B.
,
Sheetz
M. P.
(
1996
).
Targeting of Motor Proteins.
Science
271
,
1539
1544
Vallee
R. B.
,
Shpetner
H. S.
(
1990
).
Motor proteins of cytoplasmic microtubules.
Annu. Rev. Biochem
59
,
909
932
VanEtten
H. D.
(
1978
).
Identification of additional habitats of Nectria haematococca mating population VI.
Phytopathology
68
,
1552
1556
VanEtten
H. D.
,
Stein
J. I.
(
1978
).
Differential response of Fusarium solani isolates to pisatin and phaseollin.
Phytopathology
68
,
1276
1283
Verde
F.
,
Berrez
J. M.
,
Antony
C.
,
Karsenti
E.
(
1991
).
Taxol-induced microtubule asters in mitotic extracts of Xenopus eggs: Requirement for phosphorylated factors and cytoplasmic dynein.
J. Cell Biol
112
,
1177
1187
Walker
R. A.
,
Sheetz
M. P.
(
1993
).
Cytoplasmic microtubule-associated motors.
Annu. Rev. Biochem
62
,
429
451
Wilson
C. L.
,
Aist
J. R.
(
1967
).
Motility of fungal nuclei.
Phytopathology
57
,
769
771
Xiang
X.
,
Beckwith
S. M.
,
Morris
N. R.
(
1994
).
Cytoplasmic dynein is involved in nuclear migration in Aspergillusnidulans.
Proc. Nat. Acad. Sci. USA
91
,
2100
2104
Xiang
X.
,
Roghi
C.
,
Morris
N. R.
(
1995
).
Characterization and localization of the cytoplasmic dynein heavy chain in Aspergillusnidulans.
Proc. Nat. Acad. Sci. USA
92
,
9890
9894
Zhang
Z.
,
Tanaka
Y.
,
Nonaka
S.
,
Aizawa
H.
,
Kawasaki
H.
,
Nakata
T.
,
Hirokawa
N.
(
1993
).
The primary structure of rat brain (cytoplasmic) dynein heavy chain, a cytoplasmic motor enzyme.
Proc. Nat. Acad. Sci. USA
90
,
7928
7932
This content is only available via PDF.