Document S1. Four Figures, One Table, Supplemental

Current Biology, Volume 21
Supplemental Information
Distinct Roles for F-BAR Proteins
Cdc15p and Bzz1p in Actin Polymerization
at Sites of Endocytosis in Fission Yeast
Rajesh Arasada and Thomas D. Pollard
Supplemental Inventory
1. Supplemental Figures and Tables
Figure S1
Figure S2
Figure S3
Figure S4
Table S1
2. Supplemental Experimental Procedures
3. Supplemental Results
4. Supplemental References
Figure S1. Phylogenetic Analysis, Domain Organization and Localization of S. pombe FBAR Proteins
(A) Phylogenetic trees of F-BAR proteins were created with the protdist program of the PHYLIP
package. Protein sequences from the SwissProt database (accession numbers listed below) were
aligned using ClustalW and the edges and gaps were trimmed using JalView. Tree graphics were
made using the Interactive Tree Of Life (iTOL) server [10] and further enhancements to the
graphics were made with CorelDraw X4. The tree included proteins from the following species:
Dictyostelium discoideum, Dd Mgp1 (Q54GD0), Dd Mgp2 (Q55CK2), Dd Mgp3 (Q55DK5), Dd
Mgp4 (Q54QF4), DDB_G0271676 (Q75JD4), DDB_G0274695 (Q555L8); Saccharomyces
cerevisiae, Sc Rgd1 (B3LMQ1), Sc Rgd2 (B3LUG4), Sc Hof1 (B3LLT0), Sc Syp1 (P25623), Sc
Bzz1 (B3LSM9); Schizosaccharomyces pombe, Sp Rga7 (O94466), Sp Cdc15 (Q09822), Sp
Rga8 (Q09697), Sp Imp2 (Q10199), Sp Syp1 (O43059), Sp Bzz1 (Q09746), Drosophila
melanogaster, Dm Nwk (Q9VSU8), Dm Fps85D (P18106), Dm Synd (Q9VDI1), Dm Cip4
(Q8SYR8), Rattus norvegicus, Rn Pstpip1 (B0BNK4), Rn Pacsin1 (Q9Z0W5), Rn Pacsin2
(Q9QY17), Rn Pacsin3 (Q5I2Z0), Rn Gas7 (O55148), Rn Nostrin (Q5I0D6), Rn Cip4 (P97531),
Rn Fnbp1 (Q8R511), Rn Fnbp1l (Q2HWF0), Rn Fes (B2GV16), Rn Srgap2 (B5DEJ1), Mus
musculus, Mm Pacsin1 (Q61644), Mm Pacsin2 (Q9WVE8), Mm Pacsin3 (Q99JB8), Mm Pstpip1
(P97814), Mm Pstpip2 (Q99M15), Mm Fnbp1 (A2AQ41), Mm Gas7 (B1ATI9), Mm Arhgap4
(B1AUX5), Mm CIP4 (Q8CJ53), Mm Fnbp1l (Q8K012), Mm Fcho1 (Q8K285), Mm Fcho2
(Q3UQN2), Mm Fer (P70451), Mm Fchsd1 (Q6PFY1), Mm Fchsd2 (Q3USJ8), Mm Srgap1
(Q91Z69), Mm Srgap2 (Q91Z67), Mm Srgap3 (Q812A2), Homo sapiens Hs Pacsin1 (Q9BY11),
Hs Pacsin2 (Q9UNF0), Hs Pacsin3 (Q9UKS6), Hs Gas7 (O60861), Hs Fcho1 (O14526), Hs
Fcho2 (Q0JRZ9), Hs Cip4 (Q15642), Hs Fnbp1 (Q96RU3), Hs Fnbp1l (Q5T0N5), Hs Fer
(Q6PEJ9), Hs Fchsd1 (Q86WN1), Hs Arhgap4 (P98171), Hs Srgap1 (Q7Z6B7), Hs Srgap2
(O75044). The S. pombe members are highlighted in red.
(B–I) Domain organization and localization of five F-BAR proteins in S. pombe. Domain names:
F-BAR, Fer-CIP4 homology-BAR, including the Fer-CIP4 homology and α-helical sequence;
SH3, Src homology 3; GAP, GTPase activating protein; µHD, mu-Homology Domain. 3Dreconstructions of cells expressing S. pombe F-BAR proteins from stacks of 14 confocal images
taken at 0.36 μm z-intervals at 25°C.
(B) 3D-reconstructions and merged images of a mitotic cell expressing (upper panel, green)
mYFP-Cdc15p, (middle panel, red) mCFP-Myo2p. (Lower panel) merged image shows
localization of mYFP-Cdc15p with mCFP-Myo2p in the contractile ring.
(C and H) 3D-reconstructions and merged images compare the localization of (middle panels,
red) Fim1p-mCherry marking endocytic actin patches with the distinct punctate localizations of
(C) (left panel, green) Rga8p-mEGFP and (H) (left panel, green) Rga7p-mEGFP.
(D and E) 3D-reconstruction and merged images comparing the localization of (left panel, green)
Syp1p-mNFP and endocytic markers (E) End4p-mCFP (middle panel, red) and (F) Fim1pmCherry (middle panel, red).
(F) Mitotic cell expressing two F-BAR proteins (upper panel, green) mYFP-Cdc15p and (middle
panel, red) Imp2p-mCFP. (Lower panel) merged image shows Imp2p concentrated in the
contractile ring with Cdc15p.
(G) 3D-reconstructions of cells expressing (first panel, green) Rga7p-mEGFP, (second panel,
red) Rlc1p-tdTomato, and (third panel) merge. Cells at different stages of cell cycle were tilted
by 12° and represented here. (Green) Rga7p-mEGFP concentrated at the cell tips in the
interphase cells and colocalized with (red) Rlc1p-tdTomato in the contractile ring of mitotic
cells. The fourth panel shows a small region (41 X 50 pixels) at the cell center tilted by 80° to
visualize the disk-like structure of Rga7p.
(I) Domain organization of Rga9p. Scale bar, 2 μm.
(J) Growth of yeast strains depending on F-BAR proteins tagged in the genome with fluorescent
proteins at permissive (25° C) and restrictive (36° C) temperature. All strains were grown in
liquid complete (YE5S) media at 25° C, harvested in log-phase (O.D595 0.2 – 0.4) and adjusted to
2 X 107 cells/ ml. Five microliters of 10-fold serial dilutions were spotted onto YE5S plates and
incubated at 25° C or 36° C for 3-4 days.
(K) Localization of mEGFP-Cdc15p. Each panel is a maximum intensity projection of a stack of
confocal images taken at 0.36 μm intervals through (left panel) wild type cells and (right panel)
∆wsp1 mutant cells expressing mEGFP-Cdc15p. Scale bar 5 μm.
(L) Localization of Bzz1p. Each panel is a maximum intensity projection of a stack of 14
confocal images taken at 0.36 μm intervals through cells expressing (left panel) Bzz1p-mEGFP
and (right panel) mEGFP-Bzz1p. Scale bar 5 μm.
(M) FM4-64-internalization assay for endocytosis. Cells expressing the C-terminal mEGFP
tagged Bzz1p are incubated on ice for 15 min to block endocytosis, labeled with 20 μM FM4-64
on ice for 15 min, washed and resuspended in fresh ice-cold YE5S medium. Each panel
represents a single confocal plane imaged through the center of the cells on 25% gelatin pads at
(left panel) 0 min and (right panel) 30 min.
(N) Time course at 25° C of the appearance and initial constriction of S. pombe F-BAR proteins
Imp2p, Rga7p and Rga8p at the cleavage site. Separation of the spindle pole body marked with
Sad1p-mEGFP is defined as time zero.
(O and P) Time series of fluorescence micrographs at 1 s intervals of cells expressing F-BAR
proteins (O) Rga7p (green, upper panels) or (P) Rga8p (green, upper panels) tagged with mEGFP
and (red, middle panels) an actin patch marker Fim1p-mCherry. The lower panels are merged
images. Scale bar 2 µm.
Figure S2. Time Course and Quantitative Analysis of Patch Dynamics
(A–D) Time series of fluorescence micrographs of individual patches in single confocal planes at
1 s intervals in cells expressing (A) mCFP-Wsp1p and mYFP-Myo1p, (B) Crn1p-mCFP and
mYFP-Cdc15p, (C) mCFP-Wsp1p and Bzz1p-mYFP, (D) mCFP-Myo1p and mYFP-Cdc15p.
The top two rows in each panel show inverted images of a small (20 X 26 pixel) region with
single patches from the mCFP- and mYFP-images. The bottom row is a merged image with the
mCFP-images in red and mYFP-images in green. Scale bar, 0.4 μm.
(E–N) Time course of the appearance, disappearance and mean square displacements of 5
endocytic actin patch proteins tagged with mEGFP. Fluorescence intensities and positions of
individual patches were tracked in 5-optical planes at 1 s intervals for 60 s. At each time point
the numbers of mEGFP-labeled protein molecules were calculated from the total fluorescence
measured from sum-projected 5 confocal z-slices after correction for background fluorescence.
Time courses were aligned to the initiation of patch movement, defined as time zero seconds. (EH, M) Means and standard deviations of the numbers of each protein over time, with the mean
peak number of molecules ±SD in the upper right corner of each plot. (E) (●) Bzz1p (n = 15), (F)
(■) Cdc15p (n = 20), (G) (□) Myo1p (n = 18), (H) (ο) Wsp1p (n = 20), or (M) (◊) Crn1p (n =
30).
(I–L, N) Means and standard deviations of the mean squared displacements of 5 endocytic actin
patch components over time. Mean squared displacements of patches over time were calculated
from the XY coordinates on the sum-projected 2D images. (I) (●) Bzz1p (n=15), (J) (■) Cdc15p
(n=20), (K) (□) Myo1p (n=18), (L) (ο) Wsp1p (n=20), or (N) (◊) Crn1p (n=30). Error bars
represent standard deviation (SD).
Figure S3. Role of Cdc15p and Bzz1p in Endocytosis and Effects of Bzz1p Deficiency or
Cdc15p Depletion on the Time Course of Actin, Myo1p, Wsp1p and Arpc5p Accumulation
and Loss in Actin Patches
(A) Time course of the repression of mEGFP-Cdc15p protein expression during growth of the
41xnmt1megfpcdc15 strain in EMM5S medium supplemented with 2.98 μM thiamine. The
41xnmt1 promoter replaced the endogenous promoter in the native locus. The global
concentration of mEGFP-Cdc15p was calculated from the total fluorescence intensities of at least
35 cells in stacks of up to 16 z-sections spaced at 0.36 μm intervals. Imaging was done on 25%
gelatin-EMM5S pads.
(B–F) Mean square displacement of Crn1p-mEGFP patches over time calculated from the XY
coordinates on the sum-projected 2D images. (B) (ο) wild type cells (n = 10), (C) (□) ∆bzz1 cells
lacking Bzz1p (n = 12), (D) (●) cells depleted of Cdc15p by growth of a 41xnmt1cdc15 strain
with 2.98 μM thiamine (n = 13), (E) (■) ∆wsp1 cells lacking Wsp1p (n = 12), and (F) (∆) ∆myo1
cells lacking Myo1p (n = 11). (G - O) Time courses of the appearance and disappearance of (□)
mEGFP-Myo1p, (●) mEGFP-Wsp1p and (ο) ArpC5p-mEGFP in patches of cells expressing
these tagged proteins from their native loci. The data from multiple cells were aligned on the
peak values and mean values ±1 SD were plotted over time. The number in the upper right
corner of each graph is the mean peak number of molecules per patch ±1 SD.
(G–I) Molecules per patch in wild type cells: (G) (□) mEGFP-Myo1p (n = 21 patches); (H) (●)
mEGFP-Wsp1p (n = 12 patches); and (I) (ο) ArpC5p-mEGFP (n = 12).
(J–L) Molecules per patch in ∆bzz1 cells: (J) (□) mEGFP-Myo1p (n = 12 patches); (K) (●)
mEGFP-Wsp1p (n = 18 patches); and (L) (ο) ArpC5p-mEGFP (n = 8 patches).
(M–O) Molecules per patch in 41xnmt1cdc15 cells depleted of Cdc15p by growth in thiamine:
(M) (□) mEGFP-Myo1p (n = 15 patches); (N) (●) mEGFP-Wsp1p (n = 23 patches); and (O) (ο)
ArpC5p-mEGFP (n = 7).
(P–T) Experiments on GFP-actin expression. GFP-actin was expressed from the 41xnmt1
promoter in the leu+ locus in the presence of wild type levels of native actin in (P) wild-type cells
and (Q) ∆bzz1 cells lacking Bzz1p both grown in EMM5S medium at 25° C or (R) from a
3xnmt1 promoter in 41xnmt1cdc15 cells grown in EMM5S with 2.98 μM thiamine to deplete
Cdc15p.
(P–R) Time course of the appearance and disappearance of GFP-actin in actin patches in (P) (ο)
wild-type cells (Q) (●) ∆bzz1 cells lacking Bzz1p and (R) (□) 41xnmt1cdc15 cells with 2.98 μM
thiamine to deplete Cdc15p. Stacks of 5 z-sections spaced at 0.36 μm z-intervals were collected
at 1 s intervals for 60-90 s. The total number of molecules per patch was calculated from the
fluorescence intensities of at least 15 individual patches with corrections for background,
photobleaching and exposure time.
(S) Fluorescence micrographs (negative images) of maximum intensity projections of 3 confocal
planes of (left panel) wild type cells, (middle panel) ∆bzz1 cells and (right panel) 41xnmt1cdc15
cells expressing GFP-actin. Wild type and ∆bzz1 cells were grown in EMM5S while
41xnmt1cdc15 cells were grown in EMM5S with 2.98 µM thiamine. Cells were imaged on 25%
gelatin pads in EMM5S at 25° C. Scale bar, 5 µm.
(T) Measurement of GFP-actin expression by fluorescence microscopy in wild type cells and
cells lacking Bzz1p or depleted of Cdc15p. Imaging was done on 25% gelatin-EMM5S pads.
Stacks of 14 z-sections spaced at 0.36 μm intervals were collected and projected into a 2D image
using a sum intensity projection. The total concentration of GFP-actin expressed in cells was
calculated from the total fluorescence intensities of at least 10 individual cells with corrections
for cell volume, background and exposure time.
(U) 3D-reconstructions of S. pombe cells stained with BODIPY-Phalloidin. (Left panel) Wild
type cells and (right panel) wild type cells expressing GFP-actin were fixed with
paraformaldehyde and stained with BODIPY-phalloidin to visualize actin cables (white arrow
heads) and contractile rings (white arrows). 3D-reconstructions were made from stacks of 12
confocal images taken at 0.36 μm z-intervals at 25°C. Scale bar 5 µm.
(V) Time courses of the appearance and disappearance of capping protein CapBp-GFP in (ο)
wild type cells and (□) cells expressing mCherry-actin from the 41xnmt1 promoter in the leu+
locus. The fluorescence intensity of CapBp-GFP in patches was tracked in 5- optical planes at 1 s
intervals. Fluorescence intensity values for individual patches were corrected for background,
exposure time and aligned to their peaks. Error bars represent standard deviation.
Figure S4. Interaction of the SH3 Domains of Bzz1p with Wsp1p-poly (p)-VCA and its
Effect on Actin Polymerization in Actin Patches and Requirement for Myo1p forCdc15pmEGFP Association with Actin Patches in Myo1p Mutants
(A and B) Immunoblots to measure the expression of Bzz1p constructs and Cdc15 constructs.
Equal amounts of the total protein from total cellular lysates were analyzed by SDS-PAGE and
immunoblotted with anti-GFP antibodies or anti-cofilin antibodies as a loading control.
(A) Immunoblots of extracts from strains expressing Bzz1p-mYFP, Bzz1p∆SH3-mYFP or
Bzz1p∆SH3∆SH3-mYFP.
(B) Immunoblots of extracts from strains expressing Cdc15p-mEGFP or Cdc15p∆SH3-mEGFP.
(C) Quantitation of Cdc15p-mEGFP patch life times in wild type and Myo1p mutants. The
average lifetime of Cdc15p patches was measured by imaging cells expressing Cdc15p-mEGFP
in (black) wild type (n = 20), (red) myo1∆A (n = 11) and (green) myo1∆3A (n = 11) cells on 25%
gelatin pads in EMM5S at 1 s intervals.
(D) Fluorescence micrographs (negative images) at 10 s intervals of single confocal planes
through cells expressing Cdc15p-mEGFP in myo1∆23A. The cells were imaged on 25% gelatin
pads in EMM5S at 10 s intervals at 25°C. Scale bar, 2 µm.
(E) Fluorescence micrographs (negative images) of cells expressing (upper panels) Cdc15pmEGFP and (lower panels) Cdc15p∆SH3-mEGFP from the native locus imaged at 1 s intervals.
Arrows highlight the lifetime of selected patches. Cdc15p∆SH3-mEGFP appeared in actin
patches for only about 3 s. Scale bar, 1 μm.
Table S1: List of S. pombe Strains Used in this Study
Strain
FY527
FY528
TP38
TP150
Genotype
h- leu1-32 ura4-D18 his3-D1 ade6-M216
h+ leu1-32 ura4-D18 his3-D1 ade6-M210
h+ cdc15-127
h- leu1 sm902
TP190
TP190-1
h- leu1-32 ura4-D18 his3-D1 ade6-M216 myo1Δ::kanMX6
h- leu1-32 ura4-D18 his3-D1 ade6-M216 myo1Δ::kanMX6 +
pUR19-myo1+
TP194
TP194-1
h- leu1-32 ura4-D18 his3-D1 ade6-M216 wsp1Δ::kanMX6
h- leu1-32 ura4-D18 his3-D1 ade6-M216 wsp1Δ::kanMX6 +
pUR19-wsp1+
h- leu1-32 ura4-D18 his3-D1 ade6-M216 kanMX6-Pmyo1mEGFP-myo1
TP195
Source
S. Forsburg
S. Forsburg
Lab stock
=TM011, T. Toda/
M. Yanagida
Lab stock
Lab stock
Lab stock
Lab stock
Lab stock
TP199
TP219
TP226
(CB70)
TP395
JW976
JW1173
AR121
AR140
AR141
AR143
AR147
AR150
AR157
AR272
AR307
AR309
AR315
AR326
AR387
AR403
AR404
AR405
AR406
AR408
AR409
h- leu1-32 ura4-D18 his3-D1 ade6-M216 kanMX6-Pwsp1mEGFP-wsp1
kanMX6-Pmyo1-mYFP-myo1 kanMX6-Pwsp1-mCFP-wsp1
ade6-M216 leu1-32 ura4-D18 his3-D1
h- leu1-32 ura4-D18 his3-D1 ade6-M216 arc5-mEGFPkanMX6
h- leu1-32 ura4-D18 his3-D1 ade6-M216 crn1-mEGFPkanMX6
h+ cdc15-mEGFP-kanMX6 ade6-M210 leu1-32 ura4-D18
Lab stock
h- leu1-32 ura4-D18 ade6-M210 kanMX6-Pmyo2-mCFPmyo2 kanMX6-Pcdc15-mYFP-cdc15
h+ leu1-32 ura4-D18 ade6-M210 bzz1mEGFP-kanMX6
h+ leu1-32 ura4-D18 ade6-M210 kanMX6-Pcdc15-mEGFPcdc15
h leu1-32 ura4-D18 ade6-M216 bzz1mEGFP-kanMX6 fim1mCherry-NatMX6
h leu1-32 ura4-D18 ade6-M210 rga8-mEGFP-kanMX6 fim1mCherry-NatMX6
h+ leu1-32 ura4-D18 ade6-M210 rga7-mEGFP-kanMX6
fim1-mCherry-NatMX6
h leu1-32 ura4-D18 ade6-M210 imp2-mCFP-kanMX6
kanMX6-Pcdc15-mYFP-cdc15
h+ leu1-32 ura4-D18 his3-D1 ade6-M210 bzz1-mYFPkanMX6
h- leu1-32 ura4-D18 ade6-M216 rga7-mEGFP-KanMX6
rlc1-tdTomato-NatMX6
h- leu1-32 ura4-D18 his3-D1 ade6-M216 kanMX6P41xnmt1-cdc15
h- leu1-32 ura4-D18 his3-D1 ade6-M216 kanMX6P41xnmt1-cdc15 P3xnmt1-GFP-act1:leu+
h leu1-32 ura4-D18 ade6-M216 kanMX6-P41xnmt1-cdc15
arc5-mEGFP-kanMX6
h leu1-32 ura4-D18 ade6-M216 kanMX6-P41xnmt1-cdc15
crn1-mEGFP-kanMX6
h leu1-32 ura4-D18 ade6-M21X kanMX6-Pwsp1-mCFP-wsp1
bzz1-mYFP-kanMX6
h- leu1-32 ura4-D18 ade6-M210 bzz1ΔSH3- mYFP-kanMX6
h- leu1-32 ura4-D18 ade6-M210 bzz1ΔSH3 ∆SH3-kanMX6
P41xnmt1-GFP-act1:leu+
h- leu1-32 ura4-D18 ade6-M210 bzz1ΔSH3 ∆SH3- mYFPkanMX6
h- leu1-32 ura4-D18 ade6-M210 bzz1ΔSH3-kanMX6
P41xnmt1-GFP-act1:leu+
h- leu1-32 ura4-D18 his3-D1 ade6-M216 bzz1∆::ura4+
h leu1-32 ura4-D18 ade6-M21X bzz1∆::ura4+ kanMX6-
Lab stock
Lab stock
Lab stock
Lab stock
Lab stock
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
Pwsp1-mEGFP-wsp1
AR410
h leu1-32 ura4-D18 ade6-M21X bzz1∆::ura4+ kanMX6This study
Pmyo1-mEGFP-myo1
AR411
h leu1-32 ura4-D18 ade6-M21X bzz1∆::ura4+ arc5-mEGFP- This study
kanMX6
AR414
h- leu1-32 ura4-D18 his3-D1 ade6-M21X rga7-mEGFPThis study
kanMX6 sad1-GFP-kanMX6
AR417
h leu1-32 ura4-D18 ade6-M210 bzz1∆::ura4+ crn1-mEGFP- This study
kanMX6
AR419
h leu1-32 ura4-D18 ade6-M21X cdc15-127 myo1Δ::kanMX6 This study
AR423
h leu1-32 ura4-D18 ade6-M21X kanMX6-Pcdc15-mYFPThis study
cdc15 kanMX6-Pmyo1-mCFP-myo1
AR426
h leu1-32 ura4-D18 ade6-M21X kanMX6-Pcdc15-mYFPThis study
cdc15 crn1-mCFP-kanMX6
AR434
h- leu1-32 ura4-D18 his3-D1 ade6-M216 P41xnmt1-GFPThis study
act1:leu+
AR436
h leu1-32 ura4-D18 ade6-M21X bzz1∆::ura4+ P41xnmt1This study
GFP-act1:leu+
AR445
h leu1-32 ura4-D18 ade6-M21X cdc15-127 wsp1Δ::kanMX6 This study
AR448
h leu1-32 ura4-D18 his3-D1 ade6-M216 bzz1∆::ura4+
This study
wsp1Δ::kanMX6
AR450
h leu1-32 ura4-D18 ade6-M21X kanMX6-P41xnmt1-cdc15
This study
kanMX6-Pwsp1-mEGFP-wsp1
AR451
h leu1-32 ura4-D18 ade6-M216 kanMX6-P41xnmt1-cdc15
This study
kanMX6-Pmyo1-mEGFP-myo1
AR454
h leu1-32 ura4-D18 ade6-M21X myo1∆::kanMX6 crn1This study
mEGFP-kanMX6
AR455
h leu1-32 ura4-D18 ade6-M216 wsp1∆::kanMX6 crn1This study
mEGFP-kanMX6
AR458
h myo1Δ::his3+ puc1+::[pUP-myo1Δ3A ura4+] his3-D1 leu1- This study
32 ura4-D18 ade6-M21X cdc15-mEGFP-kanMX6
AR459
h myo1Δ::his3+ puc1+::[pUP-myo1ΔA ura4+] his3-D1 leu1This study
32 ura4-D18 ade6-M21X cdc15-mEGFP-kanMX6
AR460
h myo1Δ::his3+ puc1+::[pUP-myo1Δ23A ura4+] his3-D1
This study
leu1-32 ura4-D18 ade6-M21X cdc15-mEGFP-kanMX6
AR479
h leu1-32 ura4-D18 ade6-M216 syp1-mYFP-kanMX6 end4This study
mCFP-kanMX6
AR510
h- leu1-32 ura4-D18 his3-D1 ade6-M216 rga8-mEGFPThis study
kanMX6 sad1-GFP-kanMX6
AR511
h+ leu1-32 ura4-D18 his3-D1 ade6-M216 imp2-mEGFPThis study
kanMX6 sad1-GFP-kanMX6
6899
h- leu1-32 ura4-D18 ade6-M210 cdc15::cdc15ΔSH3K.L Gould
GFP/kanR
In the genotype column, "h" indicates that the strain is haploid and the mating type was not
determined as either “h+” or “h-”. M21X indicates that the ade6 allele was not determined as
either M210 or M216
Supplemental Experimental Procedures
Strain Construction, Growth Conditions, and Cellular Methods
Supplemental Table I lists the S. pombe strains used in this study. We generated all strains by
PCR-based gene targeting [11] and standard genetic methods [12]. For tagging the COOH
terminus of proteins with monomeric fluorescent proteins (FPs), DNA with the desired
homologous flanking sequences was amplified from pFA6a-FP-kanMX6 or pFA6a-FP-natMX6
plasmids. For N-terminal tagging pFA6a-kanMX6 plasmids were constructed. The pFA6akanMX6-Pcdc15-mEGFP plasmid was constructed as described for pFA6a-kanMX6-Pcdc15mYFP [2]. For the construction of pFA6a-kanMX6-Pbzz1-mEGFP 1000 base pairs of 5’ UTR
plus start codon ATG of bzz1+ were amplified and cloned into pFA6a vector digested with Bgl II
and Pac I. The plasmids were verified by sequencing. To amplify the integration cassette,
primers with 90 base pairs gene specific sequence were used. A four repeats of TCC were added
in the reverse primer for bzz1+ creating a linker of four-glycines between mEGFP and the
protein.
A ura4+ cassette replaced the entire ORF in ∆bzz1. In 41xnmt1cdc15, the 41xnmt1
promoter replaced the cdc15+ native promoter in the genome. To repress the expression of
Cdc15p, 41xnmt1cdc15 cells were grown in EMM5S supplemented with 2.98 μM thiamine.
Except where noted the native promoters controlled expression of fusion proteins from their
normal chromosomal loci. A plasmid with GFP-actin was integrated into the leu+ locus and
GFP-actin was expressed under the control of either a 3x or a 41x nmt1 promoter. All genomic
integrations were confirmed by PCR and microscopy of FPs.
Microscopy and Data Analysis
For imaging pairs of fluorescent proteins (mEGFP and mCherry or mCFP and mYFP) pairs of
images were collected at the two wavelengths before moving to the next position in Z-stacks. To
image the whole cell a stack of 14-16 z-slices at 0.36 µm steps were imaged. At each time point
we collected 5 Z-sections at 0.36-µm steps for tracking mEGFP- or mYFP-proteins in actin
patches. Patches were tracked using custom Image J plugins on images corrected for uneven
illumination and camera noise. Fluorescence intensities were tracked using concentric circles
with diameters of 0.45 µm and 0.63 µm centered on each patch. The inner circle accommodated
most of the fluorescence from the patch. The outer circle was used to measure the cytoplasmic
background fluorescence around the patch. The cytoplasmic background was calculated using
the formula, Fbackground = (FO-FI)(AI/APO-PI) [13]. FO is the fluorescence intensity in the outer
circle, FI is the fluorescence intensity in the inner circle, AI is the area of the inner circle and APOPI is the area between the perimeters of the outer and inner circles. The patch fluorescence
intensity was measured from the sum projected images by subtracting the background
fluorescence intensity from the fluorescence intensity of the inner circle, Fpatch = FI - Fbackground.
The fluorescence intensities of the patches tracked for each protein were corrected for acquisition
photo bleaching, aligned to their peaks and averaged over time. The numbers of molecules were
calculated from standard curves calibrated with seven proteins [2, 14]. Mean squared
displacements of patches over time were calculated from the XY coordinates on the sumprojected 2D images. The values obtained from each mutant were aligned in time to their peak
intensity values and averaged over time. MSD values obtained from each mutant were aligned to
the beginning of the patch movement.
Bacterial Expression Constructs
Coding sequences were amplified by RT-PCR using SuperScript III (Invitrogen) from total
S. pombe RNA isolated from wild type cells using RNeasy kit (QIAGEN). GST-Myo1pTH2SH3-CA was cloned previously [15]. For NH2-terminal GST tagging, we subcloned the
following cDNA sequences into the BamH1 and EcoR1 sites of pGEX-6P-1 (GE Healthcare):
Wsp1p poly (p)-VCA (proline-rich domain, verprolin homology motif, connecting motif and
acidic motif, nucleotides 385–1725); Bzz1pSH3SH3 (nucleotides 1561-1929); Bzz1pSH3
(nucleotides 1757-1929); and Cdc15pSH3 (nucleotides 2607-2784).
Protein Purification
Native S. pombe Arp2/3 complex was purified from a protease-deficient yeast strain [16]. GSTtagged Myo1p23A fragment was expressed in BL21(DE3)pLyS at 37°C for 4 h, purified [15]
and stored in buffer QA (10 mM Tris-HCl, pH 8.0, 1 mM EGTA, and 1 mM DTT) containing
275 mM NaCl. GST fusions to Wsp1p poly (p)-VCA (amino acid residues 129-574),
Bzz1pSH3SH3 (residues 521-642), Bzz1pSH3 (residues 586-642) and Cdc15pSH3 (residues
870-927) were expressed in ArcticExpress cells (Agilent Technologies) at 15°C and purified by
binding to glutathione-Sepharose 4B beads equilibrated with TEDABPN (10 mM Tris-HCl pH
8.0, 1 mM EGTA, 1 mM dithiothreitol, 0.02% NaN3, 1 mM benzamidine, 1 mM
phenylmethylsulfonyl fluoride, 250 mM NaCl) and eluted with 35 mM glutathione in
TEDABPN pH 7.4. GST fusion protein fractions were dialyzed against TEDABP buffer and
loaded onto a MonoQ HR 5/5 column equilibrated with the same buffer. Bound protein was
eluted using a linear gradient of 0-500 mM NaCl in TEDABP. GST was cleaved from the SH3
domains using PreScission protease (GE Healthcare) following the supplier’s protocol. SH3
domains were separated from GST on a Superdex 75 16/60 preparative size exclusion column.
Quantitative Pull-Down Assays
A range of GST-Bzz1p SH3SH3 and GST-Bzz1p SH3 concentrations [R] was immobilized on
100 µl of glutathione beads (PrepEase glutathione-agarose 4B, USB Corporation) and incubated
with a fixed concentration of soluble Wsp1p poly (p)-VCA [Ltot] at room temperature for 1 h.
After pelleting the beads with any bound ligand, concentrations of unbound ligand in the
supernatant were measured by densitometry of Coomassie blue stained SDS-PAGE. We used
KaleidaGraph (Synergy Software) to fit a binding isotherm to the dependence of the fraction of
ligand bound [LR]/[Ltot] to [Ltot] using the equation [LR]/[Ltot] = (([R] + [Ltot] + Kd) – (([R] +
[Ltot]+Kd)2 – 4 × [R] × [L])0.5)/2 × [Ltot].
Endocytosis Assays
FM4-64 assay for endocytosis was performed as described previously [17, 18]. Wild type and
∆bzz1 were grown in YE5S medium at 25°C while 41xnmt1cdc15 cells were gown at 25°C in
EMM5S supplemented with 2.98 µM thiamine. Cells were grown to an O.D595 of 0.4,
concentrated to10-fold by centrifugation. Cells were incubated at 4˚C in YE5S for 15 min prior
to the addition of the dye to block endocytosis. 20 µM of the dye was added to the cells and were
incubated for an additional 15 min at 4˚C. Cells were washed once with ice-cold YE5S, and
resuspended in ice-cold YE5S. Cells were imaged on precooled 25% gelatin pads at room
temperature every 1 min for 60 min.
Supplemental Results
Survey of Fission Yeast F-BAR Proteins
Figure 1 and S1 illustrate the domain of all seven fission yeast F-BAR proteins. In addition to the
F-BAR domains, Cdc15p and Imp2p have one C-terminal SH3 domain and Bzz1p has two SH3
domains. Rga7p, Rga8p and Rga9p each possess a C-terminal Rho GAP domain. A crystal
structure of S. cerevisiae Syp1p [1] showed that the fold of the C-terminus is similar to the cargo
binding µ homology domain (µHD) of AP2 but shares only 15% sequence identity with the AP2
µHD. Sequence identity is also low between the C-termini of S. pombe Syp1p and S. cerevisiae
Syp1.
We tagged all seven F-BAR proteins with a monomeric fluorescent protein (mNFP) in
their native loci and expressed them under the control of the endogenous promoter to determine
where each might function in fission yeast. Generally we fused the mNFP to the C-terminus of
each F-BAR protein except for Cdc15p. Cdc15p is tagged at the N-terminus as described
previously [2].
Fluorescence microscopy showed that each F-BAR protein has a unique distribution in
live fission yeast cells (Figures 1 and S1B – S1I; Table I). During interphase mNFP-Cdc15p
concentrated in actin patches [3, 4] (Figure 1A), but during mitosis mNFP-Cdc15p relocated to
equatorial nodes that condensed into a contractile ring [3, 5, 6] (Figure S1B). Imp2p-mNFP was
spread diffusely through the cytoplasm except during mitosis, when it appeared in the contractile
ring between 17 and 37 min after spindle pole body separation before the ring began to constrict
and the septum formed [6, 7] (Figures S1F and S1N). S. pombe Syp1p-mNFP concentrated in
puncta, which were distributed all around the periphery of the cells but in the highest density at
the cell tips (Figures S1D and SIE). As in S. cerevisiae [8, 9], Syp1 was immobile and remained
associated with the plasma membrane. Syp1p-mNFP colocalized in patches with the early
endocytic marker End4p-mNFP (Figure S1D), but patches with the late endocytic marker Fim1pmNFP were completely devoid of Syp1p (Figure S1E). Both Rga8p-mNFP and Rga7p-mNFP
concentrated in puncta near the plasma membrane in both interphase and mitosis, but these
puncta were distinct from actin patches tagged fimbrin or coronin (Figures S1C, S1H, S1O and
S1P, data not shown). During cell division 7 min after the spindle pole body separation Rga7p
started to concentrate around the equator in small dots distinct from nodes marked with the
Myo2p regulatory light chain, Rlc1p-tdTomato. As Rlc1p and other proteins condensed into
contractile rings, Rga7p began to accumulate and colocalize with the fully formed ring marked
with Rlc1p. Although Rga7p was not necessary for contractile ring formation, treatment of cells
with Latrunculin A showed that localization of Rga7p-mEGFP in the nascent contractile ring
depended on actin polymerization (data not shown). As the contractile ring marked with Rlc1p
constricted, more Rga7p accumulated in a disk-like structure at the interface between the
daughter cells (Figures S1G). Rga8p left the cell tips and started to accumulate at the cell center
12 min after SPB separation (Figure S1N). When expressed from either the native promoter or
the 3xnmt1 promoter in the native locus Rga9p-mYFP distributed throughout the cytoplasm
rather than concentrating in any structure during interphase or mitosis.
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