Polarization of the microtubule cytoskeleton is an early event in establishment of anterior-posterior polarity for the Drosophila oocyte. During stages 8–9 of oogenesis, when oskar mRNA is transported to the posterior pole of the oocyte, a fusion protein consisting of the plus-end-directed microtubule motor kinesin and beta-galactosidase (Kin:beta gal) similarly localizes to the posterior pole, thereby suggesting that plus ends of microtubules are pointed to the posterior. In this paper, we have substituted the motor domain of Kin:beta gal with the putative motor domain (head) from the kinesin-related protein Nod. In cells with defined microtubule polarity, the Nod:beta gal fusion protein is an in vivo minus-end reporter for microtubules. Nod:beta gal localizes to apical cytoplasm in epithelial cells and to the poles of mitotic spindles in dividing cells. In stage 8–10 oocytes, the Nod fusion localizes to the anterior margin, thus supporting the hypothesis that minus ends of microtubules at these stages are primarily at the anterior margin of the oocyte. The fusion protein also suggests a polarity to the microtubule cytoskeleton of dendrites and muscle fibers, as it accumulates at the ends of dendrites in the embryonic PNS and is excluded from terminal cytoplasm in embryonic muscle. Finally, the reciprocal in vivo localization of Nod:beta gal and Kin:beta gal suggests that the head of Nod may be a minus-end-directed motor.
References
Afshar
K.
, Barton
N. R.
, Hawley
R. S.
, Goldstein
L. S.
(1995
) DNA-binding and meiotic chromosomal localization of the Drosophila nod kinesin-like protein.
Cell
81
, 129
–138
Afshar
K.
, Scholey
J.
, Hawley
R. S.
(1995
) Identification of the chromosome localization domain of the Drosophila nod kinesin-like protein.
J. Cell Biol
131
, 833
–843
Ainger
K.
, Avossa
D.
, Morgan
F.
, Hill
S. J.
, Barry
C.
, Barbarese
E.
, Carson
J. H.
(1993
) Transport and localization of exogenous myelin basic protein mRNA microinjected into oligodendrocytes.
J. Cell Biol
123
, 431
–441
Baas
P. W.
, Deitch
J. S.
, Black
M. M.
, Banker
G. A.
(1988
) Polarity orientation of microtubules in hippocampal neurons: Uniformity in the axon and non-uniformity in the dendrite.
Proc. Nat. Acad. Sci. USA
85
, 8335
–8339
Bacallao
R.
, Antony
C.
, Karsenti
E.
, Stelzer
E.
, Simons
K.
(1989
) The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium.
J. Cell Biol
109
, 2817
–2832
Bardsley
A.
, McDonald
K.
, Boswell
R. E.
(1993
) Distribution of tudorprotein in the Drosophila embryo suggests separation of functions based on site of localization.
Development
119
, 207
–219
Berleth
T.
, Burri
M.
, Thoma
G.
, Bopp
D.
, Richstein
S.
, Frigerio
G.
, Noll
M.
, Nusslein-Volhard
C.
(1988
) The role of localization of bicoid RNA in organizing the anterior pattern of the Drosophila embryo.
EMBO J
7
, 1749
–1756
Bier
E.
, Vassin
H.
, Shepherd
S.
, Lee
K.
, McCall
K.
, Barbel
S.
, Ackerman
L.
, Carretto
R.
, Uemura
T.
, Grell
E.
, Jan
L.
, Jan
Y. N.
(1989
) Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector.
Genes Dev
3
, 1273
–1287
Brand
A. H.
, Perrimon
N.
(1993
) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.
Development
118
, 401
–415
Burton
P. R.
(1988
) Dendrites of mitral cell neurons contain microtubules of opposite polarity.
Brain Research
473
, 107
–115
Carpenter
A. T. C.
(1973
) A mutant defective in distributive disjunction in Drosophila melanogaster.
Genetics
73
, 393
–428
Chandra
R.
, Salmon
E. D.
, Erickson
H. P.
, Lockhart
A.
, Endow
S. A.
(1993
) Structural and functional domains of the Drosophila ncd microtubule motor protein.
J. Biol. Chem
268
, 9005
–9013
Cheung
H.-K.
, Serano
T. L.
, Cohen
R. S.
(1992
) Evidence for a highly selective RNA transport system and its role in establishing the dorsoventral axis of the Drosophila egg.
Development
114
, 653
–661
Clark
I.
, Giniger
E.
, Ruohola-Baker
H.
, Jan
L. Y.
, Jan
Y. N.
(1994
) Transient posterior localization of a kinesin fusion protein reflects anteroposterior polarity of the Drosophila oocyte.
Current Biology
4
, 289
–300
Desai
A.
, Mitchison
T. J.
(1995
) A new role for motor proteins as couplers to depolymerizing microtubules.
J. Cell Biol
128
, 1
–4
Driever
W.
, Nusslein-Volhard
C.
(1988
) A gradient of bicoid protein in Drosophila embryos.
Cell
54
, 83
–93
Driever
W.
, Nusslein-Volhard
C.
(1988
) The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner.
Cell
54
, 95
–104
Endow
S. A.
, Kang
S. J.
, Satterwhite
L. L.
, Rose
M. D.
, Skeen
V. P.
, Salmon
E. D.
(1994
) Yeast Kar3 is a minus-end microtubule motor protein that destabilizes microtubules preferentially at the minus ends.
EMBO J
13
, 2708
–2713
Ephrussi
A.
, Dickinson
L. K.
, Lehmann
R.
(1991
) oskar organizes the germ plasm and directs localization of the posterior determinant nanos.
Cell
66
, 37
–50
Ephrussi
A.
, Lehmann
R.
(1992
) Induction of germ cell formation by oskar.
Nature
358
, 387
–392
Erdelyi
M.
, Michon
A.-M.
, Guichet
A.
, Glotzer
J. B.
, Ephrussi
A.
(1995
) Requirement for Drosophila cytoplasmic tropomyosin in oskar mRNA localization.
Nature
377
, 524
–527
Euteneuer
U.
, McIntosh
R.
(1981
) Structural polarity of kinetochore microtubules in PtK1cells.
J. Cell Biol
89
, 338
–345
Euteneuer
U.
, Jackson
W. T.
, McIntosh
R.
(1982
) Polarity of spindle microtubules in Haemanthus endosperm.
J. Cell Biol
94
, 644
–653
Ferrandon
D.
, Elphick
L.
, Nusslein-Volhard
C.
, St Johnston
D.
(1994
) Staufen protein associates with the 3UTR of bicoid mRNA to form particles that move in a microtubule-dependent manner.
Cell
79
, 1221
–1232
Goldstein
L. S.
(1993
) With apologies to Scheherazade: tails of 1001 kinesin motors.
Ann. Review Genet
27
, 319
–351
González-Reyes
A.
, St Johnston
D.
(1994
) Role of oocyte position in establishment of anterior-posterior polarity in Drosophila.
Science
266
, 639
–642
González-Reyes
A.
, Elliot
H.
, St Johnston
D.
(1995
) Polarization of both major body axes in Drosophila by gurken-torpedo signalling.
Nature
375
, 654
–658
Grossniklaus
U.
, Bellen
H. J.
, Wilson
C.
, Gehring
W. J.
(1989
) P-element-mediated enhancer detection applied to the study of oogenesis in Drosophila.
Development
107
, 189
–200
Hawley
R. S.
, McKim
K. S.
, Arbel
T.
(1993
) Meiotic segregation in Drosophila melanogaster females: molecules, mechanisms and myths.
Ann. Rev. Genetics
27
, 281
–317
Hay
B.
, Jan
L. Y.
, Jan
Y. N.
(1988
) A protein component of Drosophila polar granules is encoded by vasa and has extensive sequence similarity to ATP-dependent helicases.
Cell
55
, 577
–587
Heidemann
S. R.
, Landers
J. M.
, Hamborg
M. A.
(1981
) Polarity orientation of axonal microtubules.
J. Cell Biol
91
, 661
–665
Horvitz
H. R.
, Herskowitz
I.
(1992
) Mechanisms of asymmetric cell division: two Bs or not two Bs, that is the question.
Cell
68
, 237
–255
Jongens
T. A.
, Hay
B.
, Jan
L. Y.
, Jan
Y. N.
(1992
) The germ cell-less gene product: a posteriorly localized component necessary for germ cell development in Drosophila.
Cell
70
, 569
–584
Kim-Ha
J.
, Smith
J. L.
, Macdonald
P. M.
(1991
) oskar mRNA is localized to the posterior pole of the Drosophila oocyte.
Cell
66
, 23
–35
Kull
F. J.
, Sablin
E. P.
, Lau
R.
, Fletterick
R. J.
, Vale
R. D.
(1996
) Crystal structure of the kinesin motor domain reveals a structural similarity to myosin.
Nature
380
, 550
–555
Kuriyama
R.
, Kofron
M.
, Essner
R.
, Kato
T.
, Dragas-Granoic
S.
, Omoto
C. K.
, Khodjakov
A.
(1995
) Characterization of a minus end-directed kinesin-like motor protein from cultured mammalian cells.
J. Cell Biol
129
, 1049
–1059
Lane
M. E.
, Kalderon
D.
(1994
) RNA localization along the anteroposterior axis of the Drosophila oocyte requires PKA-mediated signal transduction to direct normal microtubule organization.
Genes Dev
8
, 2986
–2995
Lantz
V.
, Ambrosio
L.
, Schedl
P.
(1992
) The Drosophila orb gene is predicted to encode sex-specific germline RNA binding proteins and has localized transcripts in ovaries and early embryos.
Development
115
, 75
–88
Lasko
P. F.
, Ashburner
M.
(1990
) Posterior localization of vasa protein correlates with, but is not sufficient for, pole cell development.
Genes Dev
4
, 905
–921
Lehmann
R.
, Nusslein-Volhard
C.
(1986
) Abdominal segmentation, pole cell formation, and embryonic polarity require the localized activity of oskar, a maternal gene in Drosophila.
Cell
47
, 141
–152
Lehmann
R.
, Nusslein-Volhard
C.
(1991
) The maternal gene nanos has a central role in posterior pattern formation in the Drosophila embryo.
Development
112
, 679
–691
Li
M.-G.
, McGrail
M.
, Serr
M.
, Hays
T.
(1994
) Drosophila cytoplasmic dynein, a microtubule motor that is asymmetrically localized in the oocyte.
J. Cell Biol
126
, 1475
–1494
Luo
L.
, Liao
Y. J.
, Jan
L. Y.
, Jan
Y. N.
(1994
) Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion.
Genes Dev
8
, 1787
–1802
Mahowald
A. P.
, Strassheim
J. M.
(1970
) Intercellular migration of centrioles in the germarium of Drosophila melanogaster. An electron microscopic study.
J. Cell Biol
45
, 306
–320
McDonald
H. B.
, Stewart
R. J.
, Goldstein
L. S. B.
(1990
) The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor.
Cell
63
, 1159
–1165
McGrail
M.
, Gepner
J.
, Silvanovich
A.
, Ludmann
S.
, Serr
M.
, Hays
T. S.
(1995
) Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex.
J. Cell Biol
131
, 411
–425
Nangaku
M.
, Sato-Yoshitake
R.
, Okada
Y.
, Noda
Y.
, Takemura
R.
, Yamazaki
H.
, Hirokawa
N.
(1994
) KIF1B, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria.
Cell
79
, 1209
–1220
Neuman-Silberberg
F. S.
, Schupbach
T.
(1993
) The Drosophila dorsoventral patterning gene gurken produces a dorsally localized RNA and encodes a TGFa-like protein.
Cell
75
, 165
–174
O'Kane
C.
, Gehring
W.
(1987
) Detection in situ of genomic regulatory elements in Drosophila.
Proc. Nat. Acad. Sci. USA
84
, 9123
–9127
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
Pokrywka
N. J.
, Stephenson
E. C.
(1991
) Microtubules mediate the localization of bicoid RNA during Drosophila oogenesis.
Development
113
, 55
–66
Pokrywka
N. J.
, Stephenson
E. C.
(1994
) Localized RNAs are enriched in cytoskeletal extracts of Drosophila oocytes.
Dev. Biol
166
, 210
–219
Pokrywka
N. J.
, Stephenson
E. C.
(1995
) Microtubules are a general component of mRNA localization systems in Drosophila oocytes.
Dev. Biol
167
, 363
–370
Rhyu
M. S.
, Knoblich
J. R.
(1995
) Spindle orientation and asymmetric cell fate.
Cell
82
, 523
–526
Rongo
C.
, Lehmann
R.
(1996
) Regulated synthesis, transport and assembly of the Drosophila germ plasm.
Trends in Genetics
12
, 102
–109
Roth
S.
, Neuman-Silberberg
F. S.
, Barcelo
G.
, Schupbach
T.
(1995
) cornichon and the EGF receptor signaling process are necessary for both anterior-posterior and dorsal-ventral pattern formation in Drosophila.
Cell
81
, 967
–978
Rubin
G. M.
, Spradling
A. C.
(1982
) Genetic transformation of Drosophila with transposable element vectors.
Science
218
, 348
–353
Ruohola
H.
, Bremer
K. A.
, Baker
D.
, Swedlow
J. R.
, Jan
L. Y.
, Jan
Y. N.
(1991
) Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila.
Cell
66
, 433
–449
Sablin
E. P.
, Kull
F. J.
, Cooke
R.
, Vale
R. D.
, Fletterick
R. J.
(1996
) Crystal structure of the motor domain of the kinesin-related motor ncd.
Nature
380
, 555
–559
Sharp
D. J.
, Yu
W.
, Baas
P. W.
(1995
) Transport of dendritic microtubules establishes their nonuniform polarity orientation.
J. Cell Biol
130
, 93
–103
St Johnston
D.
, Driever
W.
, Berleth
T.
, Richstein
S.
, Nusslein-Volhard
C.
(1989
) Multiple steps in the localization of bicoid mRNA to the anterior pole of the Drosophila oocyte.
Development
107
, 13
–19
St Johnston
D.
, Beuchle
D.
, Nusslein-Volhard
C.
(1991
) staufen, a gene required to localize maternal RNAs in the Drosophila egg.
Cell
66
, 51
–63
St Johnston
D.
, Nusslein-Volhard
C.
(1992
) The origin of pattern and polarity in the Drosophila embryo.
Cell
68
, 201
–219
St Johnston
D.
(1995
) The intracellular localization of messenger RNAs.
Cell
81
, 161
–170
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
Stewart
R. J.
, Thaler
J. P.
, Goldstein
L. S.
(1993
) Direction of microtubule movement is an intrinsic property of the motor domains of kinesin heavy chain and Drosophila ncd protein.
Proc. Nat. Acad. Sci. USA
90
, 5209
–5213
Tautz
D.
, Pfeifle
C.
(1989
) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos revealsa translational control of the segmentation gene hunchback.
Chromosoma
98
, 81
–85
Tetzlaff
M. T.
, Jackle
H.
, Pankratz
M. J.
(1996
) Lack of Drosophila cytoskeletal tropomyosin affects head morphogenesis and the accumulation of oskar mRNA required for germ cell formation.
EMBO J
15
, 1247
–1254
Theurkauf
W. E.
, Hawley
R. S.
(1992
) Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein.
J. Cell Biol
116
, 1167
–1180
Theurkauf
W. E.
, Smiley
S.
, Wong
M. L.
, Alberts
B. M.
(1992
) Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport.
Development
115
, 923
–936
Theurkauf
W. E.
, Alberts
B. M.
, Jan
Y. N.
, Jongens
T. A.
(1993
) A central role for microtubules in the differentiation of Drosophila oocytes.
Development
118
, 1169
–1180
Thrower
D. A.
, Jordan
M. A.
, Schaar
B. T.
, Yen
T. J.
, Wilson
L.
(1995
) Mitotic HeLa cells contain a CENP-E-associated minus end-directed microtubule motor.
EMBO J
14
, 918
–926
Walker
R. A.
, Salmon
E. D.
, Endow
S. A.
(1990
) The Drosophila claret segregation protein is a minus-end directed motor molecule.
Nature
347
, 780
–782
Wang
S.
, Hazelrigg
T.
(1994
) Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis.
Nature
369
, 400
–403
Wang
C.
, Lehmann
R.
(1991
) Nanos is the localized posterior determinant in Drosophila.
Cell
66
, 637
–647
Wilhelm
R. J.
, Vale
R. D.
(1993
) RNA on the move: The mRNA localization pathway.
J. Cell Biol
123
, 269
–274
Yang
J. T.
, Laymon
R. A.
, Goldstein
L. S. B.
(1989
) A three-domain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses.
Cell
56
, 879
–889
Yang
J. T.
, Saxton
W. M.
, Stewart
R. J.
, Raff
E. C.
, Goldstein
L. S. B.
(1990
) Evidence that the head of kinesin is sufficient for force generation and motility in vitro.
Science
249
, 42
–47
Yisraeli
J. K.
, Sokol
S.
, Melton
D. A.
(1990
) A two-step model for the localization of maternal mRNA in Xenopus oocytes: Involvement of microtubules and microfilaments in the translocation and anchoring of Vg1 mRNA.
Development
108
, 289
–298
Zhang
P.
, Knowles
B. A.
, Goldstein
L. S. B.
, Hawley
R. S.
(1990
) A kinesin-like protein required for distributive chromosome segregation in Drosophila.
Cell
62
, 1053
–1062
Zipursky
S. L.
, Venkatesh
T. R.
, Teplow
D. B.
, Benzer
S.
(1984
) Neuronal development in the Drosophila retina: monoclonal antibodies as molecular probes.
Cell
36
, 15
–26
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