Dictyostelium discoideum myosin Ik (MyoK) is a novel type of myosin distinguished by a remarkable architecture. MyoK is related to class I myosins but lacks a cargo-binding tail domain and carries an insertion in a surface loop suggested to modulate motor velocity. This insertion shows similarity to a secondary actin-binding site present in the tail of some class I myosins, and indeed a GST-loop construct binds actin. Probably as a consequence, binding of MyoK to actin was not only ATP- but also salt-dependent. Moreover, as both binding sites reside within its motor domain and carry potential sites of regulation, MyoK might represent a new form of actin crosslinker. MyoK was distributed in the cytoplasm with a significant enrichment in dynamic regions of the cortex. Absence of MyoK resulted in a drop of cortical tension whereas overexpression led to significantly increased tension. Absence and overexpression of MyoK dramatically affected the cortical actin cytoskeleton and resulted in reduced initial rates of phagocytosis. Cells lacking MyoK showed excessive ruffling, mostly in the form of large lamellipodia, accompanied by a thicker basal actin cortex. At early stages of development, aggregation of myoK null cells was slowed due to reduced motility. Altogether, the data indicate a distinctive role for MyoK in the maintenance and dynamics of the cell cortex.
REFERENCES
Adams
R. J.
, Pollard
T. D.
(1989
). Binding of myosin I to membrane lipids.
Nature
340
, 565
–568
Bastin
P.
, Bagherzadeh
A.
, Matthews
K. R.
, Gull
K.
(1996
). A novel epitope tag system to study protein targeting and organelle biogenesis in Trypanosomabrucei.
Mol. Biochem. Parasitol
77
, 235
–239
Bement
W. M.
, Mooseker
M. S.
(1995
). TEDS rule: A molecular rationale for differential regulation of myosin by phosphorylation of the heavy chain head.
Cell Motil. Cytoskel
31
, 87
–92
Bement
W. M.
, Wirth
J. A.
, Mooseker
M. S.
(1994
). Cloning and mRNA expression of human unconventional myosin-IC. A homologue of amoeboid myosins-I with a single IQ motif and an SH3 domain.
J. Mol. Biol
243
, 356
–363
Bradford
M. M.
(1976
). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem
72
, 248
–254
Browning
D. D.
, The
T.
, O'Day
D. H.
(1995
). Comparative analysis of chemotaxis in Dictyostelium using a radial bioassay method: protein tyrosine kinase activity is required for chemotaxis to folate but not to cAMP.
Cell. Signal
7
, 481
–489
Brzeska
H.
, Szczepanowska
J.
, Hoey
J.
, Korn
E. D.
(1996
). The catalytic domain of Acanthamoeba myosin I heavy chain kinase. II. Expression of active catalytic domain and sequence homology to p21-activated kinase (PAK).
J. Biol. Chem
271
, 27056
–27062
Dai
J.
, Ting-Beall
H. P.
, Hochmuth
R. M.
, Sheetz
M. P.
, Titus
M. A.
(1999
). Myosin I contributes to the generation of resting cortical tension.
Biophys. J
77
, 1168
–1176
de Hostos
E. L.
, Bradtke
B.
, Lottspeich
F.
, Guggenheim
R.
, Gerisch
G.
(1991
). Coronin, an actin binding protein of Dictyosteliumdiscoideum localized to cell surface projections, has sequence similarities to G protein beta subunits.
EMBO J
10
, 4097
–4104
de Hostos
E. L.
, Rehfuess
C.
, Bradtke
B.
, Waddell
D. R.
, Albrecht
R.
, Murphy
J.
, Gerisch
G.
(1993
). Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility.
J. Cell Biol
120
, 163
–173
Edgar
A. J.
, Knight
A. E.
, Bennett
J. P.
(1996
). Chicken myosin IB mRNA is highly expressed in lymphoid tissues.
J. Anat
189
, 451
–456
Egelhoff
T. T.
, Naismith
T. V.
, Brozovich
F. V.
(1996
). Myosin-based cortical tension in Dictyostelium resolved into heavy and light chain-regulated components.
J. Mus. Res. Cell Mot
17
, 269
–274
Evans
E.
, Yeung
A.
(1989
). Apparent viscosity and cortical tension of blood granulocytes determined by micropipet aspiration.
Biophys. J
56
, 151
–160
Fujisaki
H.
, Albanesi
J. P.
, Korn
E. D.
(1985
). Experimental evidence for the contractile activities of Acanthamoeba myosins IA and IB.
J. Biol. Chem
260
, 11183
–11189
Furch
M.
, Geeves
M. A.
, Manstein
D. J.
(1998
). Modulation of actin affinity and actomyosin adenosine triphosphatase by charge changes in the myosin motor domain.
Biochem
37
, 6317
–6326
Hammer
J. A.
III, Albanesi
J. P.
, Korn
E. D.
(1983
). Purification and characterization of a myosin I heavy chain kinase from Acanthamoeba castellanii.
J. Biol. Chem
258
, 10168
–10175
Hammer
J. A.
III, Sellers
J. R.
, Korn
E. D.
(1984
). Phosphorylation and activation of smooth muscle myosin by Acanthamoeba myosin I heavy chain kinase.
J. Biol. Chem
259
, 3224
–3229
Haugwitz
M.
, Noegel
A. A.
, Rieger
D.
, Lottspeich
F.
, Schleicher
M.
(1991
). Dictyosteliumdiscoideum contains two profilin isoforms that differ in structure and function.
J. Cell Sci
100
, 481
–489
Hayden
S. M.
, Wolenski
J. S.
, Mooseker
M. S.
(1990
). Binding of brush border myosin I to phospholipid vesicles.
J. Cell Biol
111
, 443
–451
Heintzelman
M. B.
, Schwartzman
J. D.
(1997
). A novel class of unconventional myosins from Toxoplasma gondii.
J. Mol. Biol
271
, 139
–146
Howard
P. K.
, Ahern
K. G.
, Firtel
R. A.
(1988
). Establishment of a transient expression system for Dictyosteliumdiscoideum.
Nucl. Acids Res
16
, 2613
–2623
Humbel
B. M.
, Biegelmann
E.
(1992
). A preparation protocol for postembedding electron microscopy of Dictyostelium discoideum cells with monoclonal antibodies.
Scanning Microsc
6
, 817
–825
Jung
G.
, Hammer
J. A.
III (1990
). Generation and characterization of Dictyostelium cells deficient in a myosin I heavy chain isoform.
J. Cell Biol
110
, 1955
–1964
Jung
G.
, Hammer
J. A.
III (1994
). The actin binding site in the tail domain of Dictyostelium myosin IC (myoC) resides within the glycine-and proline-rich sequence (tail homology region 2).
FEBS Lett
342
, 197
–202
Jung
G.
, Wu
X. F.
, Hammer
J. A.
III (1996
). Dictyostelium mutants lacking multiple classic myosin I isoforms reveal combinations of shared and distinct functions.
J. Cell Biol
133
, 305
–323
Kuspa
A.
, Loomis
W. F.
(1992
). Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA.
Proc. Natl. Acad. Sci. USA
89
, 8803
–8807
Lee
S. F.
, Egelhoff
T. T.
, Mahasneh
A.
, Côte
G. P.
) (1996
). Cloning and characterization of a Dictyostelium myosin I heavy chain kinase activated by Cdc42 and Rac.
J. Biol. Chem
271
, 27044
–27048
Luderer-Gmach
M.
, Liebig
H. D.
, Sommergruber
W.
, Voss
T.
, Fessl
F.
, Skern
T.
, Kuechler
E.
(1996
). Human rhinovirus 2A proteinase mutant and its second-site revertants.
Biochem. J
318
, 213
–218
Maniak
M.
, Rauchenberger
R.
, Albrecht
R.
, Murphy
J.
, Gerisch
G.
(1995
). Coronin involved in phagocytosis: Dynamics of particle-induced relocalization visualized by a green fluorescent protein tag.
Cell
83
, 915
–924
Manstein
D. J.
, Hunt
D. M.
(1995
). Overexpression of myosin motor domains in Dictyostelium: screening of transformants and purification of the affinity tagged protein.
J. Muscle Res. Cell Motil
16
, 325
–332
Manstein
D. J.
, Schuster
H. P.
, Morandini
P.
, Hunt
D. M.
(1995
). Cloning vectors for the production of proteins in Dictyosteliumdiscoideum.
Gene
162
, 129
–134
Marin
F. T.
, Rothman
F. G.
(1980
). Regulation of development in Dictyosteliumdiscoideum: IV. Effect of ions on the rate of differentiation and cellular response to cyclic AMP.
J. Cell Biol
87
, 823
–827
Mayer
B. J.
(1997
). Signal transduction: clamping down on Src activity.
Curr. Biol
7
, 295
–298
Mermall
V.
, Post
P. L.
, Mooseker
M. S.
(1998
). Unconventional myosins in cell movement, membrane traffic, and signal transduction.
Science
279
, 527
–533
Miyata
H.
, Bowers
B.
, Korn
E. D.
(1989
). Plasma membrane association of Acanthamoeba myosin I.
J. Cell Biol
109
, 1519
–1528
Murphy
C. T.
, Spudich
J. A.
(1998
). Dictyostelium myosin 25–50k loop substitutions specifically affect ADP release rates.
Biochem
37
, 6738
–6744
Neuhaus
E.
, Horstmann
H.
, Almers
W.
, Maniak
M.
, Soldati
T.
(1998
). Ethane-freezing/methanol-fixation of cell monolayers. A procedure for improved preservation of structure and antigenicity for light and electron microscopies.
J. Struct. Biol
121
, 326
–342
Noegel
A. A.
, Luna
J. E.
(1995
). The Dictyostelium cytoskeleton.
Experientia
51
, 1135
–1143
Novak
K. D.
, Peterson
M. D.
, Reedy
M. C.
, Titus
M. A.
(1995
). Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis.
J. Cell Biol
131
, 1205
–1221
Novak
K. D.
, Titus
M. A.
(1997
). Myosin I overexpression impairs cell migration.
J. Cell Biol
136
, 633
–647
Ostap
E. M.
, Pollard
T. D.
(1996
). Biochemical kinetic characterization of the Acanthamoeba myosin-I ATPase.
J. Cell Biol
132
, 1053
–1060
Ostap
E. M.
, Pollard
T. D.
(1996
). Overlapping functions of myosin-I isoforms?.
J. Cell Biol
133
, 221
–224
Pardee
J. D.
, Spudich
J. A.
(1982
). Purification of muscle actin.
Methods Cell Biol
24
, 271
–289
Pasternak
C.
, Spudich
J. A.
, Elson
E. L.
(1989
). Capping of surface receptors and concomitant cortical tension are generated by conventional myosin.
Nature
341
, 549
–551
Peterson
M. D.
, Novak
K. D.
, Reedy
M. C.
, Ruman
J. I.
, Titus
M. A.
(1995
). Molecular genetic analysis of myoC, a Dictyostelium myosin I.
J. Cell Sci
108
, 1093
–1103
Pollard
T. D.
, Korn
E. D.
(1973
). Acanthamoeba myosin. I. Isolation from Acanthamoeba castellanii of an enzyme similar to muscle myosin.
J. Biol. Chem
248
, 4682
–4690
Probst
F. J.
, Fridell
R. A.
, Raphael
Y.
, Saunders
T. L.
, Wang
A.
, Liang
Y.
, Morell
R. J.
, Touchman
J. W.
, Lyons
R. H.
, Noben-Trauth
K.
, Friedman
T. B.
, Camper
S.
(1998
). Correction of Deafness in shaker -2 Mice by an Unconventional Myosin in BAC Transgene.
Science
280
, 1444
–1447
Puta
F.
, Zeng
C.
(1998
). Blasticidin resistance cassette in symmetrical polylinkers for insertional inactivation of genes in Dictyostelium.
Fol. Biol
44
, 185
–188
Rayment
I.
, Rypniewski
W. R.
, Schmidt-Base
K.
, Smith
R.
, Tomchick
D. R.
, Benning
M. M.
, Winkelmann
D. A.
, Wesenberg
G.
, Holden
H. M.
(1993
). Three-dimensional structure of myosin subfragment-1: a molecular motor.
Science
261
, 50
–58
Rhoads
A. R.
, Friedberg
F.
(1997
). Sequence motifs for calmodulin recognition.
FASEB J
11
, 331
–340
Ridley
A. J.
(1996
). Rho: theme and variations.
Curr. Biol
6
, 1256
–1264
Rosenfeld
S. S.
, Rener
B.
(1994
). The GPQ-rich segment of Dictyostelium myosin IB contains an actin binding site.
Biochem
33
, 2322
–2328
Rosenfeld
S. S.
, Xing
J.
, Cheung
H. C.
, Brown
F.
, Kar
S.
, Sweeney
H. L.
(1998
). Structural and kinetic-studies of phosphorylation-dependent regulation in smooth-muscle myosin.
J. Biol. Chem
273
, 28682
–28690
Sellers
J. R.
, Goodson
H. V.
(1995
). Motor proteins 2: myosin.
Prot. Profile
2
, 1323
–1423
Spudich
J. A.
(1994
). How molecular motors work.
Nature
372
, 515
–518
Steck
T. L.
, Chiaraviglio
L.
, Meredith
S.
(1997
). Osmotic homeostasis in Dictyosteliumdiscoideum: Excretion of amino acids and ingested solutes.
J. Euk. Microbiol
44
, 503
–510
Stöffler
H. E.
, Bähler
M.
(1998
). The ATPase activity of Myr3, a rat myosin I, is allosterically inhibited by its own tail domain and by Ca2+ binding to its light chain calmodulin.
J. Biol. Chem
273
, 14605
–14611
Stöffler
H. E.
, Ruppert
C.
, Reinhard
J.
, Bähler
M.
(1995
). A novel mammalian myosin I from rat with an SH3 domain localizes to Con A-inducible, F-actin-rich structures at cell-cell contacts.
J. Cell Biol
129
, 819
–830
Sussman
M.
(1987
). Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions.
Methods Cell Biol
28
, 9
–29
Temesvari
L. A.
, Bush
J. M.
, Peterson
M. D.
, Novak
K. D.
, Titus
M. A.
, Cardelli
J. A.
(1996
). Examination of the endosomal and lysosomal pathways in Dictyosteliumdiscoideum myosin I mutants.
J. Cell Sci
109
, 663
–673
Titus
M. A.
(1999
). A class VII unconventional myosin is required for phagocytosis.
Current Biol
9
, 1297
–1303
Uyeda
T. Q.
, Abramson
P. D.
, Spudich
J. A.
(1996
). The neck region of the myosin motor domain acts as a lever arm to generate movement.
Proc. Natl. Acad. Sci. USA
93
, 4459
–4464
Wang
A.
, Liang
Y.
, Fridell
A.
, Probst
F. J.
, Wilcox
E. R.
, Touchman
J. W.
, Morton
C. C.
, Morell
R. J.
, Noben-Trauth
K.
, Camper
S. A.
, Friedman
T. B.
(1998
). Association of Unconventional Myosin MYO15 Mutations with Human Nonsyndromic Deafness DFNB3.
Science
280
, 1447
–1451
Wessels
D.
, Titus
M. A.
, Soll
D. R.
(1996
). A Dictyostelium myosin I plays a crucial role in regulating the frequency of pseudopods formed on the substratum.
Cell Motil. Cytoskel
33
, 64
–79
Wu
C.
, Lee
S. F.
, Furmaniak-Kazmierczak
E.
, Côte
G. P.
, Thomas
D. Y.
, Leberer
E.
(1996
). Activation of myosin-I by members of the Ste20p protein kinase family.
J. Biol. Chem
271
, 31787
–31790
Yazu
M.
, Adachi
H.
, Sutoh
K.
(1999
). Novel Dictyostelium unconventional myosin MyoK is a class I myosin with the longest loop-1 insert and the shortest tail.
Biochem. Biophys. Res. Comm
255
, 711
–716
Yeung
A.
, Evans
E.
(1989
). Cortical shell-liquid core model for passive flow of liquid-like spherical cells into micropipets.
Biophys. J
56
, 139
–149
Zhelev
D. V.
, Hochmuth
R. M.
(1995
). Mechanically stimulated cytoskeleton rearrangements and cortical contraction in human neutrophils.
Biophys. J
68
, 2004
–2014
Zhelev
D. V.
, Needham
D.
, Hochmuth
R. M.
(1994
). Role of the membrane cortex in neutrophil deformation in small pipets.
Biophys. J
67
, 696
–705
This content is only available via PDF.
© 2000 by Company of Biologists
2000
-PolarityCFP.png?versionId=5216)
