doi: 10.1242/10.1242/jcs.00159
Intracellular localization and dynamics of myosin-II and myosin-IC in live Acanthamoeba by transient transfection of EGFP fusion proteins
Hyun-Hee Kong1,* and
Thomas D. Pollard2,
1 Structural Biology Laboratory, The Salk Institute for Biological Studies, La
Jolla, CA 92037, USA
2 Department of Molecular, Cellular and Developmental Biology, Yale University,
New Haven, CT 06520-8103, USA
* Present address: Department of Parasitology, Kyungpook National University
School of Medicine, Taegu 700-422, Korea
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Fig. 1. Acanthamoeba expression vectors. (A) pUb vector has an
Acanthamoeba ubiquitin promoter (Ubp). DNAs for EGFP fusion proteins
were inserted at NcoI and XbaI restriction sites between the
promoter and the poly A signal. (B) Coding sequences for expression. PI,
profilin I; MIIt, C-terminal 256 residues of myosin-II tail; MII, full length
myosin-II; MIIm, full length myosin-II mutated at three phosphorylation sites
to alanine; MIC, myosin-IC (b, basic region; g1, GPA1 domain; s, SH3 domain;
and g2, GPA2 domain). Three phosphorylatable serines (S) in the tail piece of
pUbGMIIt and pUbGMII were replaced by three alanines (A) in pUbGMIIm.
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Fig. 2. Flow cytometry of a population of amoebas transfected with EGFP-fused
profilin-I. M1, untransfected amoeba; M2, transfected cells useful for
microscopy; M3, transfected cells with high fluorescence.
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Fig. 3. Immunoblots to measure expression of profilin-I-EGFP in
Acanthamoeba. Proteins were detected with antibodies to profilin,
actin or GFP by chemiluminescence. T, amoeba transfected with pUbPG and
selected from pools M2 and M3; C, control untransfected amoeba. Actin at 43
kDa served as the loading control for these samples. Endogenous profilins run
at 13 kDa. The 42 kDa band that reacts with both anti-profilin and anti-GFP is
the fusion protein. The band at 44 kDa that reacts with anti-GFP in both lanes
T and C is a crossreactive amoeba protein. The minor 27 kDa band that reacted
with anti-GFP but not anti-profilin is likely to be proteolytic fragment of
EGFP.
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Fig. 4. Fluorescence micrographs of live amoebas expressing (A,B) profilin-I-EGFP
and (C,D) EGFP. (A,B) Profilin-I-EGFP fills the cytoplasm and concentrates in
pseudopods. (C,D) EGFP not only fills the cytoplasm but concentrates in nuclei
except for the nucleolus. Bar, 10 µm.
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Fig. 5. Fluorescence micrographs of live amoebae expressing (A) EGFP-full
length-myosin-II or (B,C) EGFP-myosin-II tail fragment. The cells were
flattened under an agar coverslip to reduce superimposition. The direction of
movement is indicated with a white arrowhead. (A) Thick filaments labeled with
EGFP-full-length-myosin-II concentrate at the trailing edge but are also found
throughout the cytoplasm, including a few just behind the leading edge. (B,C)
Fluorescence micrographs at 0 and 8 seconds. Intense fluorescent spots
interpreted as thick filaments line up along the cell margin of the back half
and move steadily backward toward a cluster at the posterior. Small
fluorescent spots are interpreted as myosin-II minifilaments. Two white lines
serve as positional reference marks. Bar, 10 µm. See corresponding movie
online
(http://jcs.biologists.org/supplemental).
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Fig. 6. Three dimensional distribution of myosin-II filaments in a live,
unflattened amoeba expressing an EGFP-myosin-II tail fragment. Through a focus
series of fluorescence micrographs: (A) section 1 from the dorsal surface; (B)
section 3; (C) section 4; (D) section 6; (E) section 7; (F) section 10; (G)
section 13; (H) section 16, ventral surface in contact with the slide. The
cell was moving toward the upper left. Thick filaments are concentrated in the
cortex over the dorsal surface especially the back half of the cell. Bar, 10
µm.
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Fig. 7. Assembly and disassembly of myosin-II thick filaments. Two time series of
fluorescence micrographs of live, flattened amoebas expressing EGFP-myosin-II
tail fragment. Time points are given in seconds. (A) Appearance of new
filaments. When this cell changed its direction of movement from upper right
to lower left at about 20 seconds, new thick filaments (arrows) appeared
within seconds at the new tail end as the new lammellipodium (arrowheads)
emerged at lower left. The fluorescent intensity of these new filaments
increased with time. (B) Disappearance of filaments. The filament marked with
an arrow disappeared while a neighboring filament (arrowhead) moved toward the
rear (upper right). Bar, 10 µm.
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Fig. 8. Distribution of myosin-II filaments in a live, flattened amoeba expressing
constitutively active EGFP-full length myosin-II with alanine substituted for
serine at all three heavy chain phosphorylation sites. The leading edge is to
the right. Fluorescence micrographs at (A) time zero and (B) 21 seconds.
Fluorescence spots concentrated at the leading and trailing edge but also
clustered around vesicles of various sizes. Two vesicles fused between these
two time intervals (arrows). Bar, 10 µm.
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Fig. 9. Transient concentration of EFGP-myosin-IC around contractile vacuoles (CV,
black asterisk), around macropinocytic cups (MPC, white asterisk) and in
lamellipodia (LP, white arrow) in flattened amoeba expressing EGFP-myosin-IC.
These fluorescence micrographs were taken 1 second before contraction of the
contractile vacuoles and closure of the macropinocytic cups. Bar, 10
µm.
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Fig. 10. Time sequences of fluorescence micrographs of two flattened cells
expressing EGFP-myosin-IC. Fluorescence concentrates around a contractile
vacuole (CV, black asterisk) just prior to contraction and macropinocytic cups
(white asterisk) just before closure. The series of white asterisks trace the
ingestion of medium by a macropinocytic cup (MPC). Time is in seconds. Bar, 10
µm.
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Fig. 11. Behavior of EGFP-myosin-IC truncation mutants. Fluorescence micrographs of
flattened cells expressing EGFP fusions with truncated myosin-IC constructs
taken 1 second before contraction of the contractile vacuoles (CV, black
asterisks) and closure of the macropinocytic cups (MPC, white asterisks). MICs
lacks the C-terminal GPA2 region (residues I1051 to M1186). EGFP-MICg lacks
the C-terminal SH3 and GPA2 region (residues P996 to M1186). EGFP-MICb lacks
the C-terminal GPA and SH3 domains (residues L940 to M1186). EGFP-MICh lacks
the entire tail (residues N720 to M1186). Constructs with the head, basic,
GPA1 and SH3 domains concentrated around the contractile vacuoles and
macropinocytic cups. Constructs lacking the SH3 domain distributed diffusely
in the cytoplasm. Bar, 10 µm.
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© The Company of Biologists Ltd 2002
