Supplementary figure 1 A B · 8/26/2020 · Supplementary figure 2. Polarisome mutants reduced...

D

AIP577...99 bp 2027..2049 bp

2431..2453 bpCCAAAGCCGAGGCATTAGGTAAAGGTTTCGGCTCCGTAATCCATTT

1061...1083 bp

CCGACGATTCCACTCACAAGAATGGCTGCTAAGGTGAGTGTTCTTA

CCAGCGAACAACCACCTCTGGATGGTCGCTTGTTGGTGGAGACCTA

ATTTTCACTTCATTAGCCGGTGGTAAAAGTGAAGTAATCGGCCACC

+ - + - + - + -BamHIAIP5+/+

AIP5+/+, BNI1-/-

AIP5+/+, SPA2-/-

AIP5+/+, BUD6-/-

Negative m

utagenisis

E

O.D

. 600

nm

Time (h)

1.5

1.0

0.5

0.00 5 10 15 20 25

aip5∆/∆

+LatA

WT

aip5∆/∆WT

O.D

. 600

nm

Time (h)

1.5

1.0

0.5

0.00 5 10 15 20 25

bud6∆/∆bud6∆/∆ aip5∆/∆bud6∆/∆bud6∆/∆ aip5∆/∆

+LatA

bni1∆/∆bni1∆/∆ aip5∆/∆bni1∆/∆bni1∆/∆ aip5∆/∆

+LatA

O.D

. 600

nm

Time (h)

1.5

1.0

0.5

0.00 5 10 15 20 25

Time (h)

0 5 10 15 20 25

O.D

. 600

nm

1.5

1.0

0.5

0.0

spa2∆/∆spa2∆/∆ aip5∆/∆spa2∆/∆spa2∆/∆ aip5∆/∆ +LatA

F G H I

A. Conservative analysis of Bni1 among fungi species

Tree scale: 0.1

B. Conservative analysis of Bud6 among fungi species

Tree scale: 0.1

C. Conservative analysis of Spa2 among fungi species

Tree scale: 0.1

Human pathogenPlant pathogen

Supplementary figure 1

500

200

600

Supplementary figure 1. Polarisome proteins are conserved among fungi species.

(A-C) Phylogenetic tree of polarisome proteins Bni1, Bud6 and Spa2, the two highlighted species in red are S. cerevisiae

and C. albicans. (D) Four gRNA sequences with AIP5-specific target sequences as indicated. A stop codon and a BamHI

cleavage site were inserted at the location immediately following the correct mutagenesis site (highlighted by a red star)

located at the 76 bp region. (E) The negative control of CRISPR/Cas9 edited AIP5 in various C. albicans wild type and

mutant strains, where no BamHI digestion sites were inserted at the target location. (F-I) Growth curve of the indicated

polarisome C. albicans mutants that were treated with or without 1 µM latrunculin A. The data were averaged with four

technical replicates and showed with an error bar of S.D.

B

HU

arre

st c

ells

C1.0

0.8

0.6

0.4

0.2

0.0

Hyp

hae

leng

th fo

ld (%

)0-2 µm

2-3 µm

3-4 µm

4-6 µm

6-8 µm

bni1∆/∆ spa2∆/∆ bud6∆/∆WT

bni1∆/∆ aip5∆/∆ spa2∆/∆ aip5∆/∆ bud6∆/∆ aip5∆/∆aip5∆/∆

A

WTbn

i1∆/∆

spa2

∆/∆

bud6

∆/∆

bni1∆

/∆ aip5

∆/∆

aip5∆

/∆

spa2

∆/∆ aip5

∆/∆

bud6

∆/∆ aip5

∆/∆

WT

bni1∆

/∆

spa2

∆/∆

bud6

∆/∆

bni1∆

/∆ aip5

∆/∆

aip5∆

/∆

spa2

∆/∆ aip5

∆/∆

bud6

∆/∆ aip5

∆/∆


Supplementary figure 2. Polarisome mutants reduced hyphae growth length.

(A) Representative images of the indicated strain colonies with hyphae cells induced by solid agar spider plate at 37°C.

The images were shown with a magnification of 12 x, the scale bar represents 2 cm. (B) The representative fluorescence

images of actin pattern stained by phalloidin 488 in the indicated mutants of hydroxyurea (HU)-induced polarized

filaments (upper panel), and bright-field images of cell morphology (lower panel). Images were acquired from

paraformaldehyde-fixed cells after 5 hours of HU induction. The scale bar represents 2 µm. (C) Quantification of HU-

induced polarized filament length by measuring the distance between bud-neck and bud-tip. The filament length was

classified into five groups. The distribution in the percentage of each group was shown in the indicated strains. n=250

cells for each strain.

A N E A N K I G R K D I T N L E R D F R A I K Q L H N S S S E N F K E T I S N I T E Q L R K F Q E I G L E V S Q N S N R A Y M E S C N S K L S D D S D L L I T K V D D L Q D I M E E M R K D V AQ R GV G Q N K K D D D K T I K D I Q Y E L G K I K Q V H N I N R S N I N E T I F N I L R K V D N F K S L S F S A K N S S N R M Y M E K S Q T E L G D L S D T L L S K V D D L Q D V I E I M R K D V A E R R

V R V S D K Q L K H I L K D I N L A K K S L H DM T L Y I S K E R P V WK K I WE A E L D K V C E E Q Q F L N L Q D D L I Q D L Q D D I K K I E E T Y N L I E Q C S I E Q I K G A S S K R N K I V A NS Q P A K K K L E T V S K D L E N AQ A D V L K L Q E F I D T E K P HWK K T WE A E L D K V C E E Q Q F L T L Q E E L I L D L K E D L G K A L E T F D L I K L C C E E Q E K N P S R S K S N P I L P

L Y I P E P G E S L H D L K D A V L N D I V G L T P N H E S R L E A I E R A E K L R E K E R DMM K L T K F Q E E L G D F V E D K K L K R S G G I E E I E K Q R Q L R D I E N L K S S F G - - - -I - - M R P G - T F NQ V R E Q V M V A V Q S L N P D H D S R V E A I D K A E K MWE M E R K L K A S N E F D D E L E N F V G N S N L K K S G G F E E V E R I R K Q K D E A N L R A Y F G P G F T

1498

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G N S T T S S A A P P P P P P P P P P P P P P L P P I L G G N - - - - - - - - - - - - - - - - - - - N S S A A P P P P P P P P P P P A F - L - - - - - - - - - - - - - - - - - - NG S G S V I P P A PD L S T Q S S V - L S S Q P P P P P P P P P P V P A K L F G E S L E K E K K S E D D T V K Q E T T G D S P A P P P P P P P P P P P P M A L F G K P K G E T P P P P P L P S V L S S S T DG V I P P A P

P L P P P S S G R S S R S V P S T V T K S S G S A F D K I P R P K K K L K Q L HWE K I D H S Q V G N S F WN D P N T H T L V D D L M S K G I F D E I E L I F A A K E A K K L A T K K K E D L D K V TP MM P A S Q I K S - - A V T S P L L P Q S P S L F E K Y P R P H K K L K Q L HWE K L D C T - - D N S I WG T G K A E K F A D D L Y E K G V L A D L E K A F A A R E I K S L A S K R K E D L Q K I T

F L A R D I S Q Q F S I N L H A F N S F S D E E F V L K V L R C D K D V L T N P A V L D F F G K E D I V E I T N T L A R N F E P Y S T D Y K T - - - - E E I T K P E K D P N E L Q R P D R I Y L E L MF L S R D I S Q Q F G I N L HM Y S S L S V A D L V K K I L N C D R D F L Q T P S V V E F L S K S E I I E V S V N L A R N Y A P Y S T DWE G V R N L E D A K P P E K D P N D L Q R A DQ I Y L Q L M

Y N L Q H Y WK S R T R A L N V V V N Y D K D Y V E Y V K K L R L I D E A V D S I K N S K H L K G V F E I I L A V G N Y MN D S A K Q A HG F K L S S L Q R L S F M K D E K N S M T F L H Y V E K V IV N L E S Y WG S R M R A L T V V T S Y E R E Y N E L L A K L R K V D K A V S A L Q E S D N L R N V F N V I L A V G N F MN D T S K Q AQ G F K L S T L Q R L T F I K D T T N S M T F L N Y V E K I V

R T Q Y P E F L E F I N E L S C C N E I T K F S I E N I N N D C K E Y A R A I K N V Q S S I D I G N L S D V S K F H P S D R V L K A V L P A L P R A K R K A E L L L DQ A N Y T M K E F D D L M K Y FR L N Y P S F N D F L S E L E P V L D V V K V S I E Q L V N D C K D F S Q S I V N V E R S V E I G N L S D S S K F H P L D K V L I K T L P V L P E A R K K G D L L E D E V K L T I M E F E S L M H T Y

G E D P T DQ F V K N S F I S K F T D F M K D F K R V Q A E N I K R E E E L R V Y E Q R K K L L E K P K S S N NG - - - - - - - - D S N - A S DQ DG E S N E G DG G V MD S L L Q R L K A A A P T KG E D S G D K F A K I S F F K K F A D F I N E Y K K AQ AQ N L A A E E E E R L Y I K H K K I V E E Q Q K R AQ E K E K Q K E N S N S P S S E G N E E D E A E D R R A V MD K L L E Q L K N A G P A K

G E S A S A R K K A L M R K Q I L E S Q R K R T T - - - - - - - - - G - S V G S P T N V S P T R N N E S D S G V D N K D D T HG S S P L D E Q F H D S I T T Q E D R I R D D N R P P E G V A D F S N VS D P S S A R K R A L V R K K Y L S E K D N A P Q L L N D L D T E E G S I L Y S P E AMD P T A D T V - - - - - - - - - - I H A E S P T P L A T R G V MN T S E D - - - - - L P S P S K - - - T S A L

E D P E N P D V G A R A K N L L Q E L R G A D E S S S K L S D AQ R Y R Q E R L K K K S V Q I D L D E V A K N N N S E - -- - E DQ E E I S D R A R M L L K E L R G S D T P V K Q N S I L D E H - L E K L R A R K - E R S I G E A S T G N R L S F K

11227

611324

621325

1601419

1611420

2551518

2561519

3541617

3551618

4531716

4541717

5431815

5441816

6321896

6331897

6911953

Saccharomyces-cerevisiaeCandida-albicans

Bni1FH1C

Bud6C












- - - - - - - - - - - D D D D L D D L D I S P E E I R K H L E S Q P V Y I F T S L A G G - MQ V I L R S N N L A A I L Q G NG I K F E Y R D L G T D E E A K K I WK R Q A NG K T L P G V V R G D - D Y I G NWQ E I E D A N E ED S T T Q T T E Q S K K N N D K P Q D V I T T S E I R K L N E K E P V Y I Y T S L A G G G F HM I P R T N R L S T I L T A N R I P F T Y R D L G T D D E A R K V WK T F S K G R S L P G V V R G H N D L I G NWE E I E E A N E D

Y R L R E L L Y E T LY K L R E L I Y D T I

1 1001209

1011210

1111233

1110


Aip5CA

B

C


Supplementary figure 3. Conservative analysis of polarisome proteins among fungi species.

(A-C) The functional C terminal domain protein sequence alignment of Aip5, Bni1, and Bud6 between S. cerevisiae and

C. albicans.

Fluo

resc

ence

Inte

nsity

(A.U

.)

2 µM Actin (A)A+0.25 µM Aip5CA+0.5 µM Aip5CA+1 µM Aip5CA+2 µM Aip5CA+4 µM Aip5C

2 µM Actin (A)

A+20 nM Bni1FH1C

A+40 nM Bni1FH1C

A+60 nM Bni1FH1C

A+80 nM Bni1FH1C

A+100 nM Bni1FH1C

A+150 nM Bni1FH1CFluo

resc

ence

Inte

nsity

(A.U

.)

Time (s)

Fluo

resc

ence

Inte

nsity

(A.U

.)

Time (s)

2 µM Actin (A)

A+0.1 µM Bud6C

A+0.5 µM Bud6C

A+1 µM Bud6C

A+2 µM Bud6C

Fluo

resc

ence

Inte

nsity

(A.U

.)

2 µM Actin (A)A+50 nM Bni1FH1C (B)B+1 µM Aip5CB+2 µM Aip5CB+4 µM Aip5C

Fluo

resc

ence

Inte

nsity

(A.U

.)

Time (s)

2 µM Actin (A)A+50 nM Bni1FH1C (B)B+200 nM Bud6C

0 200 400 600 8000.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Flu

ores

cenc

e in

tens

ity (A

.U.) F-actin (A)

A + Aip5CA + Bni1FH1CA + Aip5C + Bni1FH1CA + Bud6CA + Bud6C + Aip5CA + Bud6C + Bni1FH1CA + Bud6C + Bni1FH1C + Aip5C

B

D

F

A C

E

0 600 1200 18000

3000

6000

9000

0 600 1200 18000

1000

2000

3000

4000

Time (s)0 600 1200 1800

0

1000

2000

3000

4000

Time (s)0 600 1200 1800

0

1000

2000

3000

4000

0 600 1200 18000

1000

2000

3000

4000

Time (s)

12085

6050

40

30

20

kDa NP-actin

0 500 1000 15000

2000

4000

6000

Fluo

resc

ence

Inte

nsity

(A.U

.)

Time (s)

2 µM Actin

2 µM NP-Actin

G

H


Time (s)0 600 1200 1800 2400

0

500

1000

1500

2000

2500

Fluo

resc

ence

Inte

nsity

(A.U

.)

2 µM Actin (A)A+1 µM Bud6C (B)B+0.5 µM Aip5CB+1 µM Aip5CB+2 µM Aip5CB+4 µM Aip5C

I

Supplementary figure 4. Polarisome proteins work synergistically to enhance actin assembly in vitro.

(A) SDS-PAGE gel of purified NP-actin (S. cerevisea actin with D286A, V287A, D288A mutations). (B) Pyrene actin

polymerization test of NP-actin. (C-E) Pyrene actin polymerization assays of an increasing concentration of Bni1FH1C,

Bud6C, and Aip5C, respectively. (F, G) Pyrene actin polymerization by an increasing concentration of Aip5C in

combination with Bni1FH1C (F) or Bud6C (G). (H) Pyrene actin polymerization by an increasing concentration of Bud6C

in combination with Bni1FH1C. (I) Pyrene actin depolymerization assay of the indicated proteins.

control

+Aip5C

+Bni1FH1C

+Bud6C

+Bud6C+Bni1FH1C

+Aip5C+Bni1FH1C

Bar

bed

end

elon

gatio

nra

te(s

ubun

its s

ec-1)

Kymograph

contr

ol

+Aip5

C

+Bni1

FH1C

+Bni1

FH1C+A

ip5C

+Bni1

FH1C+B

ud6C

+Bud

6C

+Bud

6C+A

ip5C

0 min 2 min 4 minA

B

0

5

10

15

ns

ns

ns

ns

ns

****

+Aip5C+Bud6C

+Bni1FH1C

+Bni1

FH1C+B

ud6C

+Aip5

C10

min


+Aip5C+Bud6C

Supplementary figure 5. The effect of polarisome proteins on actin filament barbed end elongation speed.

(A) The representative TIRF images of elongating actin filaments at the indicated time points. The kymographs were

generated from movies covering 10 min with 5-sec gaps in between each frame. The control actin filament was assembled

from 0.5 µM actin (10% Oregon green 488 labelled actin and 0.5% biotin-actin), and the proteins added were indicated as

follows: 20 nM Bni1FH1C; 10 nM Aip5C and 10 nM Bud6C. The scale bar represents 5 µm. (B) Quantification of actin

filament barbed end elongation speed of indicated protein combinations as shown in A. (n=40 for each sample) P-values

was determined by the one-way ANOVA, ns=not significant, ****p < 0.0001. Error bar, S.D.

F T S L A G - G MQ Y T S L A G G G F HScAip5C

Aip5C

Aip5C-G F T S L A G G GMQ

A

B

0 600 1200 1800 24000

500

1000

1500

Time (s)

Fluo

resc

ence

Inte

nsity

(A.U

.)

Actin 2 µM (A)A+Aip5C-G 2 µMA+Aip5C-G 4 µMA+Aip5C-G 8 µM

C

250

75

5037

kDa Aip5C-G


Supplementary figure 6. Aip5C mutants and activation in actin assembly.

(A) Protein sequence comparison of the functional loop domain between C. albicans and S. cerevisiae. (B) The protein

sequence of Aip5C-G where the Glycine was inserted. The purified Aip5C-G was shown in SDS-PAGE. (C) The bulk

actin assembly was tested by pyrene assay with increasing concentrations of Aip5C-G.

1545 a.a. 1733 a.a.Bni1C

CaB

ni1

Dis

orde

red

Tend

ency

Amino acid

A B C

D

1 2

3 4

5 6

7 8

E F

G H

I J

K L

1200

1300

1400

1500

1600

1700

0.0

0.5

1.0 FH2 domain C terminus

250755037

kDa Bni1C

25


Supplementary figure 7. Initial Bni1C-Bud6C-Aip5C complex structure placements for MD simulations

(A) The recombinant Bni1C (residues 1545-1733 a.a.) was purified and shown in SDS-PAGE. (B) The intrinsically

disordered tendency plot of protein Bni1FH1COOH by using IUPRED2A. While Bni1C is highly disordered, the FH2

domain is predicted to be well folded as the disorder tendency score is lower than 50%. (C, D) The initial structure in MD

simulations for Bni1C (C) and Bud6C (D). (E-L) The initial eight sets of Bni1C-Bud6C-Aip5C complex placements,

where Bni1C was placed along the x-axis with N terminus on the left side, and Aip5C and Bud6C were assigned into four

directions (right, left, up, and down) relative to Bni1C.

Simulation

-1650

-1600

-1550

-1500

Ros

etta

sco

re

A

1

6

B D

6

6

2

2

2

4

4

5

5

5

7

7

1

7

Gibbs Energy Landscape

PC2

PC1

0 14.2G (kJ/mol)

C

1


Time (ps)

RM

SD(n

m)

0 5×104 1×105 1.5×105 2×1050

1

2

3

4

Time (ps)

RM

SD(n

m)

0 5×104 1×105 1.5×105 2×1050

1

2

3

4

5

Time (ps)

RM

SD(n

m)

0 5×104 1×105 1.5×105 2×1050

1

2

3

4

5

Time (ps)

RM

SD(n

m)

0 5×104 1×105 1.5×105 2×1050

1

2

3

4

Time (ps)

RM

SD(n

m)

0 5×104 1×105 1.5×105 2×1050

1

2

3

4

5

Time (ps)

RM

SD(n

m)

0 5×104 1×105 1.5×105 2×1050

1

2

3

4

5 4

Supplementary figure 8. The RMSD plots and Rosetta scoring of the stable tri-protein complex from MD

simulations

(A) The RMSD plots of six groups (Simulation 1, 2, 4, 5, 6, 7) that maintain stable complex conformations after MD simulations. Simulation 3 and 8 complexes were excluded because of the full or partial detachments of three proteins after MD simulations. (B) Free energy landscape of the first two Principle components（PC1 and PC2) for the tri-complexes (Simulation 1). The energy scale representing the stable (Blue) to least stable (Red) structures ranging from 0 to 14.2 kcal/mol. (C) The Rosetta scores of the six sets of tri-protein complex after MD simulations. (D) The conformations of the most stable assembly of tri-protein complex for each set (including conformation 1 in figure 6C).

Supplementary Table 1. Yeast strains used in this study.

Strain name Genotype Source

YMY2045

(BWP17) ura3/ura3 his1::hisG/his1::hisG arg4::hisG/arg4::hisG (1)

YMY2046 BWP17 spa2Δ::ARG4/spa2Δ::FRT (2)

YMY2047 BWP17 bni1Δ::ARG4/bni1Δ::HIS1 (3)

YMY2048 BWP17 bud6Δ::ARG4/bud6Δ::HIS1 (3)

YMY2049 BWP17 aip5Δ:: NATR/aip5Δ:: NATR This study

YMY2050 BWP17 spa2Δ::ARG4/spa2Δ::FRT aip5Δ:: NATR/aip5Δ:: NATR This study

YMY2051 BWP17 bni1Δ::ARG4/bni1Δ::HIS1 aip5Δ:: NATR/aip5Δ:: NATR This study

YMY2052 BWP17 bud6Δ::ARG4/bud6Δ::HIS1 aip5Δ:: NATR/aip5Δ:: NATR This study

Supplementary Table 2. Oligonucleotide primers used in the study.

SgAIP5-F ATTCTTGTGAGTGGAATCGTGTTTTAGAGCTAGAAATAGCAAGTTAAAA

SgAIP5-R ACGATTCCACTCACAAGAATCGGATCCAAATTAAAAATAGTTTACGCAA

GT

RtAIP5-F TCCATCTTTGGTTGGATCTTCCATTAGATCATAAGGATCCACGATTCCACT

CACAAGAAT

RtAIP5-R TGATATCTTTGGAAGATTTGTATTTTTCTGGGTGGATATAATTCTTGTGAG

TGGAATCGT

VerAIP5-F GACATTGAAGAGCGTTTAG

VerAIP5-R TTTACCTCCAACATTATCAG

1. Wilson, R. B., Davis, D., and Mitchell, A. P. (1999) Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. Journal of bacteriology 181, 1868-1874

2. Wang, H., Huang, Z. X., Au Yong, J. Y., Zou, H., Zeng, G., Gao, J., Wang, Y., Wong, A. H. H., and Wang, Y. (2016) CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth. Molecular microbiology 101, 250-264

3. Li, C. R., Wang, Y. M., De Zheng, X., Liang, H. Y., Tang, J. C. W., and Wang, Y. (2005) The formin family protein CaBni1p has a role in cell polarity control during both yeast and hyphal growth in Candida albicans. Journal of cell science 118, 2637-2648

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Supplementary figure 1 A B · 8/26/2020 · Supplementary figure 2. Polarisome mutants reduced...

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