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SUPPLEMENTARY ONLINE MATERIAL
Supplementary methods
Strains, plasmids and expression constructs
The Ustilago maydis strains AB33GT, FB2, , AB33∆Myo5, AB33∆Myo5rKin1,
AB33∆Kin1, SG200G3Mcs1, AB33G3Dyn2, SG200G3Mcs1_mChsso1,
FB2crgHAMcs1HN and SG200G3Mcs1ΔΜΜ were described previously
(Schuster et al. 2011b; Banuett & Herskowitz 1989; Lehmler et al., 1997;
Schuchardt et al., 2005; Lenz et al., 2006; Treitschke et al., 2010). The
genotypes of all strains used in this study are summarized in Table 1. The
following plasmids were generated:
pomChTub1. The plasmid potefGFPTub1 (Steinberg et al, 2001) was digested
with NcoI and NdeI to remove the GFP gene and replace it with mCherry
gene resulting in plasmid pomChTub1.
pHomChTub1. To obtain this plasmid the carboxin resistance cassette was
removed form plasmid pomChTub1 by digesting with NotI. It wars replaced by
the hygromycin resistance cassette resulting in plasmid pHomChTub1.
poLifeactG. Actin was visualised by a 17 amino acid peptide, representing
the actin-binding region of ABP140p in S. cerevisiae
(MGVADLIKKFESISKEE; Riedl et al, 2008) fused to eGFP (Lifeact-GFP).
LifeAct-GFP plasmid was constructed through in vivo recombination in the
Saccharomyces cerevisiae strain DS94 (MATα, ura3-52, trp1-1, leu2-3, his3111, and lys2-801 (Tang et al., 1996) following published procedures
(Raymond et al., 1999). The otef promoter and the gfp gene were amplified
from plasmid poGRab5a (Schuster et al, 2011a) by using chimeric primers
which contain 17 amino acid lifeact sequence with Neurospora crassa-codon
optimised nucleotides (ATG GGC GTC GCT GAC CTC ATC AAG AAG TTC
GAG TCC ATC TCC AAG GAG GAG; Berepiki et al, 2010). The PCR
amplified products were recombined into the BamH1 and HindIII digested
plasmid pNEBcbx-yeast (Schuster et al, 2011a) in Saccharomyces cerevisiae
resulting in poLifeActG. Plasmid poLifeactG was linearized with AgeI for
homologous integration at the succinate dehydrogenase locus of strain AB33
resulting in strain AB33LifeActG.
pChs5G3 and pChs6G3. To visualize chitin synthase 5 (Chs5) and chitin
synthase 6 (Chs6), two additional copies of gfp were introduced into the
plasmids pChs5GFP and pChs6GFP (Weber et al. 2006) resulting in
pChs5G3 and pChs6G3, respectively. Both the plasmids were digested with
SspI and integrated homologously into the chs5 and chs6 locus of strain FB1
resulting in FB1Chs5G3 and FB1Chs6G3 respectively.
pcrgKin3G105E. Plasmid pcrgKin3G105E (Wedlich-Söldner et al., 2002) was
digested with SspI and integrated at the succinate dehydrogenase locus of
strain AB33Mcs1G3 resulting in AB33Mcs1G3_rKin3rigor. To visualize the early
endosomes (EE), plasmid pomChRab5a (Schuster et al., 2011a) was
digested with ScaI and integrated ectopically in to the genome of
AB33Mcs1G3_rKin3rigor resulting in AB33Mcs1G3_rKin3rigor_mChRab5a.
pHomChRab5a. The Nourseothricin resistance cassette in the plasmid
pomChRab5a (Schuster et al., 2011a) was replaced by a hygromycin
resistance cassette, resulting in the plasmid pHomChRab5a. The plasmid
pHomChRab5a was linearized and integrated ectopically in to the AB33∆kin3
strain (Schuster et al., 2011b) resulting AB33∆kin3_mChRab5a. Plasmid
pn3GMcs1 (Treitschke et al., 2010) was linearized and integrated in to the
succinate dehydrogenase locus resulting AB33∆kin3_mChRab5a_G 3Mcs1.
pNcrgKin1rigor. The plasmid pHcrgKin1G96E (Straube et al., 2006) was
digested with XbaI and BamHI to remove the hygromycin phosphotransferase
gene resistance cassette and parts of the back bone. A pNEB193 vector (New
England Biolabs, Ipswich, USA) containing the nourseothricin resistance
cassette was digested with BglII and SpeI and the resulting 4163 bp fragment
was fused to the 6664 bp fragment derived from pHcrgKin1G96E. The
resulting plasmid pNcrgKin1rigor was linearized by digestion with NotI and
integrated ectopically into the genome of AB33Mcs1G3 and A33G3Myo5,
resulting in strains AB33G3Mcs1_rKin1rigor and A33G3Myo5_rKin1rigor.
pCcrgKin1rigor. To obtain plasmid pCcrgKin1rigor the plasmid pNcrgKin1G96E
was digestet with NotI to remove the carboxin resistance cassette and replace
it by the nourseothricin resistance cassette. This plasmid was transformed
into strain AB33G3Dyn2 (Lenz et al., 2006) resulting in strain AB33 G3Dyn2_
Kin1rigor.
pG3Myo5 and pmCherry3Myo5. The plasmids pG3Myo5 and
pmCh3Myo5were generated through in vivo recombination in the yeast S.
cerevisiae using strain DS94 (MAT , ura3-52, trp1-1, leu2-3, his3-111, and
lys2-801 (Raymond et al., 1999). Fragments with 30 bp homology to the
upstream and downstream of the sequence stretch of interest were amplified
by PCR using 35 cycles and purified from the agarose gel. In order to obtain
the yeast - E. coli shuttle vector, a 2680 bp fragment containing the yeast
URA3 marker and 2µm ori amplified from plasmid pEYA2 (Invitrogen, Paisley,
UK) and was cloned in to the plasmid pNEBhyg (Brachmann et al,. 2001),
which was linearized by SacI. The resulting plasmid pNEBhyg-yeast
contained the ampicilin resistance cassette, an E. coli origin of replication, and
the hygromycin phosphotransferase gene resistance cassette. A 782 bp
fragment covering the left flank, 988 bp of the myo5 promoter and 1010 bp of
the myo5 gene was cloned in to SacI and SphI digested plasmid pNEBhygyeast along with either the 708 bp mCherry gene or with the 717 bp gfp gene,
both amplified by PCR and both fused to 30 bp of the myo5 promoter and 30
bp of the myo5 gene. The resulting plasmids pGMyo5 and pmChMyo5 were
digested with BsrGI and two additional copies of gfp or mCherry were
introduced as BsrGI fragments. To integrate the resulting plasmid
pmCh3Myo5-hyg into strain AB33Mcs1G3, the hygromycin
phosphotransferase gene resistance cassette was exchanged with
nourseothricin resistance cassette. The plamids pG3Myo5 and pmCh3Myo5
were digested with SspI and integrated homologously into the myo5 locus of
strains A33 and AB33Mcs1G3 resulting A33G3Myo5 and AB33
Mcs1G3_Ch3Myo5 respectively.
pmCh3Mcs1.To visualize the Mcs1, a 832 bp fragment upstream of the mcs1
promoter, the nourseothricin resistance cassette, 958 bp mcs1 promoter
followed by mCherry and 1100 bp of the start of the mcs1 gene sequence
were cloned into a cloning vector resulting in plasmid pmChMcs1. The
plasmid pmChMcs1was digested with BsrGI and two additional copies of
mCherry were introduced as BsrGI fragments resulting in the plasmid
pmCh3Mcs1. The plasmid pmCh3Mcs1 was digested with XhoI and integrated
homologously into the mcs1 locus of strain AB33G3Myo5 resulting
AB33G3Myo5_mCh3Mcs1.
pcrgMyo5rigor. This plasmid was obtained by cloning of a 3549 bp fragment of
the crg-promotor (Bottin et al., 1996) into p123 via NotI/NcoI. The full-length
myo5-gene including ~1000bp of the 3´UTR was amplified using primers 5´CAACCCGGGATGGCACCCGCACCTGCC-3´and 5´CAACCCGGGGAACTGAGTCTGAATCCAGACTCCAG-3´ and ligated into
p123-Pcrg-vector via NdeI and EcoRI. At the N-terminus of myo 5 a HA-tag
was cloned via NcoI/NdeI obtaining the plasmid p123-Pcrg-HA-myo5. The
rigor-mutation G183E was introduced using QuikChange® Site-Directed
Mutagenesis Kit (Stratagene, La Jolla, USA) and primers 5´CGGTGAGTCCGGTGCCGAGAAGACGGTATCCGCAAAGTAC-3´ and 5´GTACTTTGCGGATACCGTCTTCTCGGCACCGGACTCACCG-3´.
pn3GMcs1rigor. This plasmid was generated using plasmid pn3Mcs1 and the
QuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, USA) and
primers 5´-CGGAGACACCTCAAGCGAAAAGAGCGAAGTTCGTCGTC-3´
and 5´-GACGACGAACTTCGCTCTTTTCGCTTGAGGTGTCTCCG-3´ to
generate the G113E-point mutation.
pET15b-Mcs1Hrigor. Plasmid pET15b-Mcs1Hrigor was generated by
amplification of mcs1H (aa 1-878) and subsequent cloning into the pET15bvector (Novagen, Madison, USA) via NdeI and BamHI. The point mutation
was generated using the QuikChange® Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, USA).
Growth conditions
U. maydis liquid cultures were grown overnight in complete medium
containing 1% (w/v) glucose (CMglucose; Holliday, 1974), shaking at 200
revolutions per minute at 28ºC. AB5Dyn2ts_Mcs1_3G was grown permissive
temperature (22 ºC) in CMglucose and shifted to restrictive conditions (32 ºC) for
2 hours. For induction of the crg-promoter in strains AB33Mcs1G3_rKin1rigor
AB33 G3Dyn2_ Kin1 rigor, AB33 G3Myo5_ Kin1 rigor and
AB33Mcs1G3_rMyo5rigor, cells were grown in CM-glucose medium to an OD600
≈ 0.5 and transferred into CM-medium containing 1% (w/v) arabinose as sole
carbon source (CMarabinose) and incubated for the indicated times at 28ºC,
shaking at 200 revolutions per minute (rpm). Strain AB33∆Myo5rKin1 was
grown in complete medium containing 1% (w/v) arabinose. To repress the
crg-promoter the cells were transferred into CM-medium containing 1% (w/v)
glucose and incubated for 12 hours at 28ºC, shaking at 200 revolutions per
minute (rpm).
Laser-based epifluorescence-microscopy
Cells were placed onto a 2% agar cushion and observed using a IX81
motorized inverted microscope (Olympus, Hamburg, Germany), equipped with
a PlanApo 100x/1.45 Oil TIRF objective (Olympus, Hamburg, Germany) and a
VS-LMS4 Laser-Merge-System with solid state lasers (488 nm/70 mW and
561 nm/70 mW, Visitron System, Munich, Germany). For photo-bleaching
experiments a 405 nm/60 mW diode laser, which was attenuated by a ND 0.6
Filter, resulting in 15 mW output power, coupled into the light path by a OSI-IX
71 adaptor (Visitron System, Munich, Germany) and controlled by a UGA-40
controller (Rapp OptoElectronic GmbH, Hamburg, Germany) and a VisiFRAP
2D FRAP control software for Meta Series 7.5.x (Visitron System, Munich,
Germany). Simultaneous observation of red and green fluorescent protein
fluorescence was done using a Dual-View Microimager (Photometrics,
Tucson, USA) equipped with a dual line beam splitter (z491/561, Chroma,
Rockinham, USA), an emission beam splitter (565 DCXR, Chroma,
Rockinham, USA), an ET-Bandpass 525/50 (Chroma, Rockinham, USA) and
a BrightLine HC 617/73 (Samrock, Rochester, USA). Images were acquired
using a Photometrics CoolSNAP HQ2 camera (Roper Scientific, Germany).
All parts of the system were under the control of the software package
MetaMorph (MDS Analytical Technologies, Winnersh, UK).
Quantitative analysis of fluorescent intensities and motility
All measurements were carried in 14 bit images using the software
MetaMorph. Intensity measurements of G3Mcs1 and mutated versions and
images of medium-sized budded cells were taken at an exposure time of 200
to 250 ms. Analysis of signal intensities was done in the growth region by
measuring average intensities at the plasma membrane. Measured values
were corrected for cytoplasmic background. From the corrected values the
mean average intensity value was calculated. For velocity and frequency
measurements, image series of 75 to 150 frames at 200 ms were taken. The
frequency of G3Mcs1 motility in motor-mutant strains was determined by
counting signals that crossed a line in the middle of the mother cell over time.
All measurements were related to control cells grown at identical conditions.
Velocities of G3Mcs1 and G3Myo5 signals were measured in kymographs
using MetaMorph. All statistical analyses were done using the software Prism
4 (GraphPad, La Jolla, CA, USA).
FRAP-based secretion assays
CHSs secretion rates were determined after pre-treatment with DMSO,
Benomyl or Latrunculin A for 15 min (see details above) and placed 2% agar
cushions containing the respectively inhibitor. A reverence image was taken
at 100% of the 488 nm observation laser at an exposure time of 150 ms
followed by a 100 ms light pulse using a 405 nm laser at 70 % laser power
(beam diameter 30) to bleach the whole bud. An image was acquired directly
after bleaching and after 15 or 20 minutes. The integrated intensity of the all
CHS accumulating at the bud tip was measured in all three images and the
relative recovery determined by comparison of these images. To determine
secretion of CHSs the percentage of the second image was subtracted form
the third one.
Cells of strains SG200G3Mcs1 and SG200G3Mcs1∆MM were placed onto a 2%
agar cushion. The whole bud or one flank of the mother cell was photobleached by a 100 ms light pulse using a 405 nm laser (60 mW) at 70 % laser
power. An image was acquired directly after the bleaching and a image series
of 75 plains after 5 minutes there taken using 100% of the 488 nm
observation laser at an exposure time of 250 ms. Stable insertion of individual
signals was confirmed in kymographs using MetaMorph. The number of
inserted signals per 1 µm plasma membrane and per 5 minutes was
determined.
Actin co-sedimentation assay
His-Mcs1H and His-Mcs1Hrigor were expressed using the “TNT® T7 Quick
Coupled Transcription/Translation” Kit (Promega, Madison, USA) and
plasmids pET15b-Mcs1HN and pET15b-Mcs1HNrigor, according to the
manufacturer’s instructions. Prior to co-sedimentation protein containing cell
extracts were centrifuged for 1 h at 100,000g at 4°C and supernatants were
used. F-actin was polymerized an actin-binding kit (Cytoskeleton, Denver,
USA) according to the manufacturer’s instructions. F-actin was sedimented for
1 h at 100,000g at 4°C and the pellet resuspended in stabilizing buffer (20 mM
Tris-HCl, pH 8,0, 5 mM MgCl2 and 2 µM Phalloidin), followed by incubation for
4 h at 4°C. Supernatants containing His6-Mcs1H and His6-Mcs1Hrigor were
incubated with 3 µM F-actin in the presence of 7 mM MgCl2 and 0.5 U
apyrase (NEB) or 5 mM ATP, respectively. After incubation of 15 min at 20°C
samples were centrifuged for 1 h at 100,000g at 4°C. Supernatant and pellet
fractions were analyzed by Western blotting using an anti-His antibody
(1:10000; Sigma-Aldrich, Taufkirchen, Germany).
In vitro motility assays
Partial purification of G3Mcs1-containing membranes.
Cells of strains SG200G3Mcs1 or SG200G3Mcs1∆MM were grown overnight
and protoplasts were prepared as previously described (Schulz et al, 1990).
Protoplasts were sedimented by centrifugation at 0.6 g and resuspended in
100 µl AB-buffer (4°C, containing 25 mM imidazole, 25 mM KCl, 4 mM MgCl2,
1 mM EGTA and 2 mM DDT, pH 7.4). The cells were disrupted by adding
100-150 mg of 0.40- 0.60 mm glass beads, followed by 3 min vibration on a
IKA Vibrax VXR (IKA-Werke, Staufen, Germany). Cell debris and organelles
were removed by centrifugation at 5000 g for 5 min at 4°C. For single
molecule assays, 50 μl of crude cell extract was mixed with 50 μl 1 M KCl and
kept on ice for 10 minutes, followed by centrifugation at 40 000 rpm in a 40
000 Beckman TL100 centrifuge through 100 μl of 15 % (w/v) sucrose cushion
in AB buffer with protease inhibitors. The pellet washed twice with AB buffer
with protease inhibitors and resuspended in 40 μl AB buffer and stored on ice
until used in the single molecule in vitro assays.
TIRF optical arrangement. A microscope flowcell arrangement was created
using a microscope slide (25 mm x 75 mm; “Superfrost”, Menzel-Glaser
GmbH & Co., Braunschweig, Germany) and a #1 coverslip (22 mm x 40 mm;
Menzel-Glaser). The coverslip was fixed orthogonally across the central
region of the slide using two strips of double-sided tape (“Permanent Double
Sided Tape”:#34-8507-7367-3, Scotch 3M, Bracknell, Berkshire, UK) so that a
channel (10 mm x 25 mm x 100 μm deep, i.e. 25 μL volume) was formed
between slide and coverslip and the coverslip ends projected by about (7.5
mm) either side of the slide edge. The inverted microscope arrangement
(TE2000, Nikon, Kingston, Surrey, UK) used in these experiments requires
that the coverslip forms the lower surface of the flowcell (i.e. adjacent to the
microscope objective lens), whilst the slide is uppermost and fixed firmly to
the microscope stage. The projecting ends of the coverslip, on either side of
the slide, enabled experimental solutions to be added at one side using a
pipette and removed from the other using filter paper. The sample region was
illuminated using a totally internally reflected 532 nm laser beam (Nd:YVO4,
frequency-doubled laser, Suwtech LDC-1500, Shanghai, China) or a 488 nm
argon ion laser beam (Omnichrome, Melles Griot, CVI Laser Ltd, Leicester,
UK) using a high numerical aperture microscope objective lens (Nikon Apo
TIRF ×100, NA 1.45). The laser was focused at the back aperture of the
objective using an external lens and the incident angle of the incoming laser
beam was adjusted using an external mirror arrangement in order to achieve
total internal reflection. Fluorescence emitted from the coverslip surface was
collected through the same objective lens and passed through a dichroic and
bandpass filter (560DRLP & 595AF60 for rhodamine or 505DRLP & 535AF45
for GFP; Omega Optical, Brattleboro, VT, USA) to be imaged onto an
intensified CCD camera (PTI IC-300, PTI Inc., Ford, West Sussex, UK).
Spatial calibration was performed using a graticule which gave 83 nm in both
x and y directions per square pixel. Video data was captured at 25 frames per
sec (40ms per frame) using a Picolo Pro-3 (Euresys Inc., Itasca, IL USA)
frame grabber card and proprietary software (GMpicolo16.exe available at
http://www.nimr.mrc.ac.uk/gmimpro/). Break-through of the red (rhodamine)
signal into the green (eGFP) fluorescence channel depended upon how
brightly labeled the actin filaments were labeled (see below for details). This
was corrected by setting the threshold level for the GFP signal detection
above a critical value, which was determined empirically for each video record
(see below).
Experimental solutions and buffers. All chemicals were sourced from SigmaAldrich (Gillingham, Dorset, UK). The flowcell surface coating procedure and
motility assays (see below) were conducted using an assay buffer, AB
consisting of: 25 mM imidazole-HCl, 25 mM KCl, 4 mM MgCl2, 1 mM EGTA,
pH 7.4; AB+ as AB but with 2 mM ATP added. Oxygen-scavenger buffer,
OS/AB consisted of 0.02 mg/ml catalase, 0.05 mg/ml glucose oxidase, 3
mg/ml glucose, 0.5 mg/ml BSA and 2 mM DTT made up in either degassed
AB or AB+. Solutions were stored in hypodermic syringes, fitted with 23 gauge
needles, to reduce oxygen contamination and syringes containing the oxygenscavenger buffer mix were warmed to 30 oC for 5 min before use to ensure
efficient enzymatic removal of oxygen.
Acto-myosin interaction assay. The principle of the acto-myosin interaction
assay is to immobilize filamentous actin on a microscope coverslip and then
observe myosin binding to and moving along the immobilized actin tracks.
The flowcell was assembled (as above) and biotin-BSA (0.1 mg/ml) in AB was
added and incubated for 5 min. The flowcell was then washed 5 times with AB
and neutravidin (0.5 mg/ml) in AB was added and incubated for 5 min. The
flowcell was then washed 5 times with AB and rhodamine-phalloidinstabilized, biotinylated filamentous actin was added to the flowcell (20 nM
monomeric actin concentration) in AB. The actin filaments were made using
equimolar, biotin-labeled actin (Cytoskeleton Inc, Denver, CO, USA);
unmodified actin and rhodamine phalloidin. The flow cell was then washed
with OS/AB to remove unbound actin and the surface was visualized using
TIRFM with 532 nm laser excitation and rhodamine filter set. This method
gave about 10 filaments (of about 10μm length) bound for each 40x40 μm
square field of view. GFP-tagged M17 (5μl of myosin stock mixed with 45 μl of
either OS/AB or OS/AB+) was introduced into the flowcell and GFP
fluorescence monitored by TIRFM using 488 nm laser excitation and GFP
filter set. During the experiment, the actin filament positions and GFP-tagged
myosin-17 were observed by switching between laser excitation wavelengths
and respective filter sets.
ATP depletion from experimental solutions. To remove contaminant Mg.ATP
from the myosin 17-containing stock solution, 1μl of 1 mg/ml apyrase
(equivalent 0.06 Units of enzyme activity) was incubated with 99 μl of myosin
stock solution at 23°C for 30 minutes. This gave complete conversion of the
contaminant ATP to AMP with no detectable ADP or ATP. The reaction was
monitored by strong anion exchange (SAX) high pressure liquid
chromatography. Briefly, 2 μl samples collected at different time points were
loaded onto the SAX column, followed by isocratic elution using 0.4 M
ammonium phosphate, pH 4.0 with 5% acetonitrile. Using a flow rate of 1
mg/ml; AMP, ADP and ATP were resolved as separate peaks and
concentrations were estimated from the peak absorption measured at 254 nm
wavelength.
Video data analysis of G3Mcs1 binding to actin filaments. Binding of eGFPtagged Mcs1 to immobilized rhodamine-phalloidin labeled actin filaments was
determined by first creating a “ghost” image of the actin filament positions
using the red filter set and then overlaying this image onto subsequent video
images of the eGFP labeled myosin, recorded using the green (eGFP) filter
set. Positions of myosin-17 molecules were analyzed in movie sequences of
1000-1500 frames, taken at 25 fps. Break-through of the actin (rhodamine)
signal into the myosin (eGFP) fluorescence channel was corrected by
threshold the eGFP signal above a critical value. Positions of myosin-17
molecules were plotted as a kymograph centered at the actin filament position
on the image.
Supplementary figures and figure legends
Supplementary Figure S1 Effect of cytoskeleton inhibitors on F-actin and MTs.
In the presence of 30 µM Benomyl all MTs are disassembled (Fuchs et al., 2005).
However, LifeAct-GFP still decorates F-actin cables. Disruption of F-actin by 10 µM
Latrunculin A (Fuchs et al, 2005) did not affect the MT organization. Thus, both
filamentous systems do not depend on each other. Bar represents micrometers.
Supplementary Figure S2 Localization of 3xGFP fused to Mcs1, the myosin-17 in
U. maydis. The fusion protein (green) concentrates in the plasma membrane (red,
mCherry-Sso1) of growing buds. Image series shows stationary G3Mcs1 signal
(arrowhead) beneath the plasma membrane. Time is given in seconds; bars
represent micrometers.
Supplementary Figure S3 Kymographs showing motility behavior of secreted
G3Mcs1 within the plasma membrane in control cells (Control) and cells treated with
the F-actin inhibitor Latrunculin A (+LatA). Images are contrast inverted.
Supplementary Figure S4 Importance of early endosome motility for
secretion of Mcs1.
(A) Apical concentration of G3Mcs1 in a kinesin-3 null mutant. Note that
kinesin-3 delivers EEs to the growth region (Wedlich-Söldner et al., 2002). Bar
represents 5 micrometers.
(B) Images showing the localization of mCherry-Rab5a labeled EEs and
G3Mcs1 in a mutant expressing a rigorously binding kinesin-3 mutant protein
(Kin3rigor; Wedlich-Söldner et al., 2002). While EEs are not moving
(arrowheads; compare 0 min and 2 min), G3Mcs1 still concentrates at the
growth region, suggesting that secretion of the CHS is not impaired. Bar
represents 2 micrometers.
(C) Bar chart showing secretion rates of G3Mcs1 in control cells, kinesin-3 null
mutants (Kin3) and kinesin-3 rigor (Kin3rigor). The error probability P was
obtained from a one-way ANOVA test and indicates no significant difference
between all three data sets.
Supplementary Figure S5 Motility of G3Mcs1 along microtubules.
Image series shows G3Mcs1 (green) moving along microtubules, labeled with
mCherry fused to tubulin (red). Time is given in seconds; bar represents a
micrometer.
Supplementary Figure S6 Motility of Mcs1-carrying secretory vesicles in the
absence of the myosin-17 motor domain (MM).
(A) The mutant protein G3Mcs1MM (MM) no longer concentrates at the
growth region, suggesting an essential role of the motor domain in secretion
of the chitin synthase region of Mcs1. Note that this confirms previous results
in hyphal cells (Treitschke et al, 2010). Bars are given in micrometers.
(B) Kymographs showing motility of the truncated protein G3Mcs1MM in
photo-bleaching experiments. Time is given in seconds, distance is given in
micrometers. The image was contrast inverted.
(C) Frequency, velocity and run-length of G3Mcs1 (control) and G3Mcs1MM
(MM) vesicles. Values are given in mean±SEM, sample size is indicated.
Note that no statistic differences were found (P>0.05).
Supplementary Figure S7 Co-sedimentation of a recombinant motor head of
Mcs1 containing a point mutation predicted to cause rigorous binding to Factin.
(A) Domain organization of the rigor-mutant protein.
(B) Western blot showing that most recombinant rigor mutant protein cosediments with F-actin in the presence of ATP.
Supplementary movie legends
Movie S1 The cytoskeleton in growing yeast-like cells of U. maydis.
3D-maximum reconstructions from Z-axis stacks after deconvolution. F-actin
is labelled by Lifeact-GFP, MTs are labelled by GFP-tubulin. Bars
represent micrometers.
Movie S2 Anterograde motility of G3Mcs1 (green) in a photo-bleached bud
(edge in blue). Bar represents micrometers; elapsed time is given in
seconds and milliseconds.
Movie S3 Insertion of G3Mcs1 (green) into the plasma membrane, labelled
with mCherry-Sso1 (red). Bar represents micrometers; elapsed time is given
in seconds and milliseconds.
Movie S4 Behaviour of G3Mcs1 (green) at the plasma membrane (red) in a
bud of a yeast-like cell. Bar represents micrometers; elapsed time is given in
seconds and milliseconds.
Movie S5 Motility of G3Mcs1 (green) along microtubules labelled with a
fusion protein of mCherry and the tubulin Tub1 (red). Bar represents
micrometers; elapsed time is given in seconds and milliseconds.
Movie S6 Motility of G3Mcs1in control cells, temperature-sensitive dynein
mutants (Dyn2ts), kinesin-1 null mutants (Kin1) and mutants expressing a
rigorously binding Kin1-mutant protein (Kin1rigor). Images were contrast
inverted. Bar represents micrometers; elapsed time is given in seconds and
milliseconds.
Movie S7 Motility of G3Mcs1 (green) independently of microtubules labelled
with a fusion protein of mCherry and the tubulin Tub1 (red). Bar represents
micrometers; elapsed time is given in seconds and milliseconds.
Movie S8 Anterograde motility of a fusion protein of 3xGFP and Myo5, the
class V myosin of U. maydis. Note the peripheral flow of signals towards the
growing bud. Bar represents micrometers; elapsed time is given in seconds
and milliseconds.
Movie S9 Anterograde motility of a fusion protein of 3xGFP and Myo5 in a
daughter bud. Note the peripheral flow of signals towards the growing bud.
Bar represents micrometers; elapsed time is given in seconds and
milliseconds.
Movie S10 Co-imaging of G3Myo5 and mCherry3Mcs1. Centrally moving
Mcs1 signals (red) are not co-localizing with the peripheral myo5 (green; left
cell). Peripheral moving Mcs1 co-localizes with myosin-5 (arrowhead in right
cell. Note that the images were misaligned by on pixel to better visualize colocalization of G3Myo5 and mCherry3Mcs1.
Movie S11 Motility of G3Mcs1 in mutant cells that express a mutated Myo5
protein that rigorously binds to F-actin. Note that cells the apical
concentration of Myosin-17 is gone and "pearl-string"-like signals appear at
the cell periphery (arrowheads). Bar represents micrometers; elapsed time
is given in seconds and milliseconds.
Movie S12 Predicted structure of the myosin-motor domain. The folding of
the motor domain of Mcs1 was predicted by comparison with published
structures of 4 myosins. Myosin 5a from chicken (Myo5a) and the myosin17 (Mcs1) share a similar fold.
Movie S13 Binding of G3Mcs1 to F-actin in vitro. G3Mcs1 signals (green)
transiently co-localize with F-actin (red), but no motility occurred. Bar
represents micrometers; elapsed time is given in seconds and milliseconds.
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