Supplementary Information Titles - Nature Research€¦ · Supplementary information Phospholipid dependent regulation of the motor activity of myosin X Nobuhisa Umeki1, Hyun Suk

Supplementary Information Titles

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Journal: Nature Structural & Molecular Biology

Article Tracking Number:

NSMB-A25334C Article Title: Phospholipid dependent regulation of the motor activity of

myosin X

Corresponding Author:

Mitsuo Ikebe

Supplementary Item & Number (add rows as needed)

Title or Caption

Supplementary Figure 1

SDS-PAGE of the purified myosin X constructs.


Cross-linking of full-length and the tail-truncated myosin X.


Representative negatively stained fields and averaged images of myosin X constructs.


Tail-induced inhibition of M10∆GTD ATPase activity.


Effect of ionic strength on the tail-induced inhibition of the actin-activated ATPase activity of M10∆GTD under EGTA conditions.


Amino acid sequence alignment of the PH domains.


Myosin X lipid overlay assay.


Models for the regulation of the motor activity and the filopodia induction/cargo transportation activity of myosin X.

Supplementary Methods

Nature Structural & Molecular Biology: doi:10.1038/nsmb.2065

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Supplementary information

Phospholipid dependent regulation of the motor activity of myosin X

Nobuhisa Umeki1, Hyun Suk Jung2, Tsuyoshi Sakai1, Osamu Sato1, Reiko Ikebe1, and Mitsuo Ikebe1

1Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA01655, 2Division of Electron Microscopic Research, Korea Basic Science Institute, 52 Eoeun-dong, Daejeon 305-333, Korea.

N.U., H.S.J., and T.S. equally contributed to the work.Correspondence should be addressed to this author ([email protected]).

Supplementary Figure 1-8Supplementary Methods


200

1169766

45

29

2014

1 2 43

Supplementary Figure 1. SDS-PAGE of the purified myosin X constructs. Lane 1, M10full; lane 2, M10∆GTD; Lane 3, M10M5cc : Lane 4, M10IQ0. Molecular masses are indicated in the left.

(kDa)


200

11697

(kDa)

Reaction Time (min)

0 15 30 0 15 30

M10∆GTD M10M5cc66

b

Supplementary Figure 2. Cross-linking of full-length and the tail-truncated myosin X. (a) Cross-linking of the globular tail truncated myosin X (M10∆GTD) and M10M5cc containing the coiled-coil domain of myosin V. Molecular masses (kDa) are indicated at left. (b) Cross-linking of full-length myosin X. M10full LZ having a Leucine Zipper motif at the C-terminal end was used as a control. Cross-linking was done as described in Materials and Methods. The samples before and after cross-linking were analyzed by SDS-PAGE followed by Western blot using anti-Flag antibodies. The position of dimer and monomer, respectively, are indicated at right.

Reaction Time (min)

0 10 20 0 10 3030 40 20 40

M10full M10full LZ

Dimer

Monomer

a


M10full(High salt + ATP)

a b

c d M10full(Low salt - ATP)

M10tail

M10IQ0

M10∆GTD

M10∆GTD + M10tail

M10fullSupplementary Figure 3. Representative negatively stained fields and averaged images of myosin X constructs. (a, b) Negatively stained fields of M10full in following condition: (a) M10full in 500 mM Na acetate and in the presence of ATP; (b) M10full in 50 mM Na acetate and in the absence of ATP (APO). Black and white arrowheads in (a, b) indicate the appearance of M10full, appearing narrower shapes. (c) Field of the tail construct in 50 mM Na acetate under the final concentration of 30 nM. Black arrows in (c) point to the appearances of the tail construct. Inset image in (c) shows averaged image of M10tail construct consisting of 8 particles (among 178 processed particles in total). Note that the tail construct appears very flexible shapes along most of its length. (d) Averaged images of M10IQ0, M10∆GTD, M10∆GTD interacting with M10tail construct, and M10full (c.f. head-only structure (upper row panels; M10IQ0 and M10∆GTD) and head-tail structure (lower row panels;M10∆GTD+M10tail and M10full)). Each average consists of 20-50 images. Asterisk marked averages were selected and presented in Fig. 3e. 50nm and 20nm scale bars apply to fields (a-c with a inset average in c) and averaged images (d), respectively.

20nm


b

c

Supplementary Figure 4. Tail-induced inhibition of M10∆GTD ATPase activity. (a) Actin depen-dence of the inhibition by exogenous tail domain (M10tail) under EGTA conditions. Open circles, in the absence of M10tail; closed circles in the presence of 0.5 µM M10tail. (b) Tail-induced inhibition of the forced dimer construct of M10M5cc. Effect of exogenous tail domain (M10tail) on the actin-activated ATPase activity of M10M5cc under EGTA (Closed circles) and pCa4 (Open circles) was measured in presence of 20µM actin. (c) Effect of exogenous tail on the basal ATPase activity of M10∆GTD. 0.5 µM M10tail was used. Values are mean with SE from 3 independent experiments.

Actin

-act

ivat

edAT

Pase

act

ivity

(%)

Basa

l ATP

ase

Activ

ity (s

-1)

ATPa

se a

ctiv

ity (s

-1)

Actin (µM)

M10tail (µM)

a


Supplementary Figure 5. Effect of ionic strength on the tail-induced inhibition of the actin-activated ATPase activity of M10∆GTD under EGTA conditions. Open circles, absence of M10Tail; Closed circles, presence of M10Tail. Open triangles, the ratio of ATPase activity in the presence and absence of M10Tail. 0.5 µM tail and 20 µM actin were used.


AKT1 K E G W L H K R W R P R Y F L L KG E Y I K NBTK

X65687 MmL E S I F L K R L29788 Mm

Hs

M10 PH2 domain K Q G W L H K K

21a 1b 3M10 PH domain

G G G S S T L S R R N W K K R W F V L R

β1 β2Loop

*1 2 1 5

* *1 2 3 1 1 2 3 3

GAP1M GRP1 R E G W L L K L

S Q Q K K K T S P L N

G G R V K T W K R R W F I L

F K K R L F L L

PDK1

GAB1 C S G W L R K S P P E K K L K R Y A W K R R W F V L RE N N L I L K M G P V D K R K G L F A R R R Q L L L

ITK L E E Q L I K K S Q Q K R R T S P S N F K V R F F V L Q08881AF001871BAB32975U43885AF017995

MmMmHsHs

Accession Sp

BtU55042

CONSENSUS GxxKxASP

K L

K E G E M Y K R A Q G R T R I G K K N F K K R W F C L

IQCoiled-coil

Motor Domain PEST PH MyTH4 FERM1 2052

M10 PH1 domain H S F L Y M K G G G L M N S W K R R W C V L K BtU55042M10 PH3 domain V R G W L H K E L K K R W F V L TV K N S P K M S S L K BtU55042

x R x R x F x L

Supplementary Figure 6. Amino acid sequence alignment of the PH domains. Sequence similar-ity between the PH domain of myosin X and other PH domains of PtdIns(3,4,5)P3-binding proteins is shown. The asterisks represent the mutated conserved residues in the PH2 domain of myosin X in this study. GenBank accession No. and species (Sp) of each sequence are shown on the right. Bt: Bos Taurus, Mm: Mus musculus, Hs: Homo sapiens. A consensus sequence derived from the con-served residues is also shown by shaded box. The PH1 and PH2 sequences are shown at the bottom.


Control

PI PI(3,4)

P2PIP

3PI(4

,5)P2

(pmol)

M10PH-FERMM10full

162

54

18

6

2

(pmol)

162

54

18

6

2

162

54

18

6

2

M10PH-FERM(K1215A/R1231C)

Control

PI PI(3,4)

P2PIP

3PI(4

,5)P2

Control

PI PI(3,4)

P2PIP

3PI(4

,5)P2

(pmol)

M10PH2 M10PH3100

50

25

a

b(pmol)

Supplementary Figure 7. Myosin X lipid overlay assay. (a) M10full, M10PH-FERM or M10PH-FERM(K1215A/R1231C) was overlaid onto a nitrocellulose membrane spotted with lipid vesicles. These proteins bound to lipids were detected using anti-Flag antibody (sigma). (b) GST-M10PH2 and GST-M10PH3, respectively were overlaid onto a nitrocellulose membrane spotted with lipid vesicles. These proteins bound to lipids were detected using anti-GST-antibody (sigma). PI, phosphatidylinosi-tol; PI(3,4)P2, phosphatidylinositol-3,4-bisphosphate; PIP3, phosphatidylinositol-3,4,5-triphosphate; PI(4,5)P2, phosphatidylinositol-4,5-bisphosphate.

PI(3,4)

P2PIP

3PI(4

,5)P2

PI(3,4)

P2PIP

3PI(4

,5)P2


PIP3Monomeric M10 (inhibited form)

Actin filament

Filopodia Cell body

Dimeric M10 (active form)

PEST

PHMyTH4

FERM

PEST PH MyTH4 FERM

Motor domain

Motor domain

Inactive

Active

PIP3

PIP3

a

b

Supplementary Figure 8. Models for the regulation of the motor activity and the filopodia induction/cargo transportation activity of myosin X. (a), Motor activity of myosin X is inhibited by the binding of the tail to the head domain. This occludes the dimer formation compatible region in the tail. PtdIns(3,4,5)P3 (PIP3) binding to the PH domain disrupts the tail/head interaction, which abolishes the tail-induced inhibition of the motor activity. The activated conformation is compatible for the dimer formation. (b), Myosin X encounters PtdIns(3,4,5)P3 (PIP3) at the leading edge, thus forming the dimer of active conformation. Activated myosin X induces actin structural rearrangement to form the base of filopodia, and transports its cargo complex to the filopodial tips.


Supplementary Methods

Materials. Restriction enzymes and modifying enzymes were purchased from New England

Biolabs (Beverly, MA) unless indicated otherwise. Pfu Ultra High-Fidelity DNA polymerase

was purchased from Stratagene (La Jolla, CA). Oligonucleotides were synthesized by

Invitrogen (Carlsbad, CA). Vector plasmid pFastBacHT was purchased from Invitrogen.

Vector plasmid pGEX and Glutathione-Sepharose 4B resin was purchased from GE

Healthcare. Anti-FLAG M2 affinity gel, FLAG peptide, phosphoenol pyruvate, 2,4-

dinitrophenyl-hydrazine, pyruvate kinase, and L-Glutathione were from Sigma. (St. Louis,

MO). Actin was prepared from rabbit skeletal muscle according to Spudich and Watt 1.

Recombinant calmodulin was expressed in Escherichia coli and purified as described

previously2.

Cell Culture and transfection. COS7 cells (ATCC) were cultured with Dulbecco's modified

Eagle's medium (D-MEM) containing 10% (v/v) fetal bovine serum (FBS) at 37˚C and 5%

CO2. Transient transfections were performed with GeneJammer Transfection Reagent

(STRATAGENE) according to the manufacture’s instructions. Transfected plasmid DNA was

purified using Qiagen mini-or maxi-prep columns. The cells were fixed at 14 h after

transfection, and the number of filopodia, which showed the tip localization of GFP-signal,

was counted for randomly selected cells.

Protein lipids overlay assay. Prepared liposome containing 2–162 pmol of phosphoinositides

dissolved in methanol/chloroform/water (2:1:0.8 volume ratio) was spotted onto Protran

Nitrocellulose Membranes (Schleicher & Schuell) and dried at room temperature for 1 h. The

membrane was blocked in 5% fat-free BSA with gentle rocking in blocking buffer (50 mM

Tris (pH 7.5), 150 mM NaCl, 0.1% (v/v) Tween-20) at 4 °C overnight. Membranes were

incubated with gentle rocking in the blocking buffer containing 1 nM of the indicated myosin

X constructs at 4 °C overnight. The membrane was washed 10 times over a period of 1 h in

Tris-buffered saline (TBS) containing 0.05% (v/v) Tween-20, and then incubated for 1 h with


an anti-Flag antibody (sigma) or anti-GST antibody (sigma). After washing, the membranes

were incubated with horseradish peroxidase-conjugated anti-mouse antibody (Bio-Rad) for 1

h at room temperature. After washing, signals were detected by Super Signal West Pico

(Pierce Chemical).

Pull-down assay. M10IQ0 and M10PH-FERM with FLAG tag were incubated in buffer

containing 20 mM HEPES pH7.5, 50 mM KCl, 1 mM MgCl2, 0.1 mM DTT, 1mM ATP and 1

mM EGTA at 25˚C for 10 min, then was further incubated with 50 µl of anti-FLAG antibody

resin for 90 min at 4°C. The beads were washed extensively, proteins eluted in Laemmli

sample buffer, and resolved by SDS-PAGE. Densitometric analysis was performed using the

NIH ImageJ version 1.38 software.

Confocal microscopy. Fluorescence images were viewed with a Leica DM IRB laser

scanning confocal microscope controlled by Leica TCS SP II systems (Leica Microsystems)

equipped with a Plan-Apochromat 60x 1.40 NA oil immersion objective (Leica). The images

were processed using Photoshop software (Adobe).

Preparation of liposomes. PC(phosphatidyl choline), PS(phosphatidyl serine),

PI(phosphatidylinositol) and PtdIns(4,5)P3 (phosphatidylinositol, 4,5-bisphosphate) were

purchased from Avanti Polar Lipids. PtdIns(3,4)P3 (phosphatidylinositol, 3,4-bisphosphate)

and PtdIns(3,4,5)P3 (phosphatidylinositol, 3,4,5-triphosphate) were purchased from Enzo Life

Sciences. These lipids were mixed in chloroform in a glass vial and evaporated using dry

nitrogen in a fume hood. The lipids were dissolved in 5mM HEPES (PH7.5) and were

subjected to four freeze and thaw cycles. After sonication using a water-bath type sonicator

(Bransonic model 2510) in ice cold water for 20 min, the liposomes were stored in the dark at

4°C and used within 1 week.

1. Spudich, J.A. & Watt, S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem 246, 4866-71 (1971).


2. Ikebe, M. et al. A hinge at the central helix of the regulatory light chain of myosin is critical for phosphorylation-dependent regulation of smooth muscle myosin motor activity. J Biol Chem 273, 17702-7 (1998).


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Supplementary Information Titles - Nature Research€¦ · Supplementary information Phospholipid dependent regulation of the motor activity of myosin X Nobuhisa Umeki1, Hyun Suk