Download Supplementary material Recruitment of a myosin

Supplementary material
S1
Recruitment of a myosin heavy chain kinase to actin-rich
protrusions in Dictyostelium
Paul A. Steimle, Shigehiko Yumura, Graham P. Côté, Quint G. Medley,
Mark V. Polyakov, Brian Leppert, and Thomas T. Egelhoff
Current Biology 1 May 2001, 11:708–713
Supplementary materials and methods
Figure S1
DNA methods and cell maintenance
We fused GFP to the amino-terminus of the MHCK A coding region by
using the vector pDXA-GFP2 [S1]. An EcoRI site was engineered by
PCR at codon 2 of the MHCK A coding region. This allowed the entire
MHCK A gene to be inserted at the corresponding site of pDXA-GFP2.
The resultant construct relied on the native MHCK A codon for translation
termination. We subjected all PCR-derived segments to DNA sequence
analysis to confirm the correct final sequence. The FLAG epitope-tagged
version of MHCK A was generated by transferring the insert from the
GFP fusion into the vector pTX-FLAG [S1]. Both constructs were introduced into the Ax2 cell line via electroporation as described previously
[S2]. Dictyostelium cells were maintained in HL5 [S3] in plastic petri
dishes. Cells expressing GFP or FLAG fusions to MHCK A were grown
in medium supplemented with 10 ␮g/ml G418 (Geneticin; Gibco). For
chemoattractant response studies with GFP-MHCK A, cells were differentiated to the aggregation stage as described previously [S4].
Detergent-insoluble cytoskeleton assays
Triton-resistant cytoskeleton analysis was performed with aggregation
stage cells. Cells (4 ⫻ 107 cells/ml) in suspension culture were first
incubated for 2 hr in starvation buffer (20 mM MES [pH 6.8], 0.2 mM
CaCl2, 2 mM MgSO4) and then were exposed to 100 nM cAMP (final
concentration) that was applied from a syringe pump every 6 min for
4–6 hr. We then brought the cells to a basal stimulatory state by adding
caffeine (100 ␮M final concentration) to the cell slurry and then incubating
them for 30 min. Subsequently, the cells were stimulated with 100 ␮M
cAMP, and at indicated times after stimulation, aliquots of cells were
lysed by the addition of an equal volume of 20 mM TES (pH 6.8), 1%
Triton-X-100, 2 mM MgCl2, and 5 mM EGTA. Triton-resistant cytoskeletal
pellets were collected by centrifugation, suspended in 2⫻ sample buffer
and subjected to SDS-PAGE and Western blot analysis with an antiMHCK A polyclonal antibody [S5]. The relative amount of actin and
myosin associated with pellets was determined by densitometric analysis
of the scanned Coomassie-stained gels. Likewise, the amount of pelletassociated MHCK A was quantified by densitometry of developed Western blots. The MHC null cell line HS1 [S6] was used for similar experiments assessing MHCK A distribution behavior in the absence of myosin.
Microscopy
Conventional immunomicroscopy was performed with either Ax2 parental
cells or with FLAG-MHCK A cells. All cells were fixed in ⫺15⬚C ethanol
containing 1% formaldehyde for 5 min and then rinsed with PBS. The
fixed cells were incubated with a previously described anti-MHCK A
monoclonal antibody [S7] or with the commercial anti-FLAG epitope
monoclonal antibody M2 (Sigma). Actin staining was performed with
tetramethylrhodamine-labeled phalloidin. After 30 min of rinsing with
PBS, FITC-labeled goat anti-mouse IgG antibodies were incubated for
1 hr. For Figures 1a and 1c, cells were processed for microscopy by
the agar overlay method [S8]. For all other fixed-cell microscopy images
(Figure 2a in the main text and Figure S1), no agar overlay was used.
In a number of additional experiments, similar localization has been observed regardless of which fixation method was used. Immunostained
samples were observed with a Zeiss Axiovert microscope/ confocal laser
system. An oil immersion Plan-Neofluor 100x objective lens (NA 1.3)
was used for image collection.
Further assessment of cell responses to chemoattractant in the
presence of latrunculin A (Lat-A). It was noted that GFP-MHCK A
acquired a granular cytosolic appearance when cells were stimulated
with cAMP in the presence of Lat-A. To determine whether this
might represent active recruitment to cytosolic structures of some sort,
we tested the behavior of a construct that has the actin binding
domain (ABD) of ABP-120 fused to GFP (GFP-ABD-120). This
construct was previously shown to have a persistent cortical
localization due to its association with cortical F-actin [S9]. In our
tests, GFP-ABD-120 was cortically localized in developed Ax2 cells
and remained so when cells were stimulated with cAMP (left panels).
Pretreatment with Lat-A to disassemble cortical F-actin caused GFPABD-120 to become diffuse and cytosolic in distribution (upper right
panel). As with GFP-MHCK A, stimulation with cAMP in the presence
of Lat-A induced a granular fluorescence pattern, suggesting
reorganization of the GFP-ABD-120 protein into particulate
structures of some sort. Ax2 cells expressing GFP alone did not display
a granular appearance when they were stimulated with chemoattractant
(not shown). We speculate that the granular appearance of these two
distinct GFP constructs (ABD-120 versus MHCK A) upon
chemoattractant stimulation may represent loci within the cells where
transient F-actin polymerization may be occurring briefly despite the
presence of Lat-A.
S2
Supplementary references
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