Materials

Restriction enzymes and modifying enzymes were purchased from New England Biolabs (Beverly, MA). Oligonucleotides were synthesized by Invitrogen Co. (Beijing, China). The GoScript Reverse Transcription System Kit was from Promega. Ni-nitrilotriacetic acid-agarose (Ni-agarose) was purchased from Qiagen (Hilden, Germany). Glutathione (GSH)-Sepharose 4 Fast Flow was from GE Healthcare. Anti-Flag M2 affinity agarose and HPR conjugated anti-Flag M2 antibody were from Sigma Co. (St. Louis, MO). FLAG peptide (DYKDDDDK) was synthesized by Augct Co. (Beijing, China). Pentabromopseudilin (PBP) was from AdipoGen Life Sciences (Switzerland). Blebbistatin (-) was from Selleck (Houston, USA). Nocodazole was from Shanghai yuanye Bio-Technology Co., Ltd. Sodium arsenite was from BeNa Culture Collection (Beijing, China). Cycloheximide (CHX) was from Sigma-Aldrich (St. Louis, MO, USA).

Proteins

All Myo5a constructs in this study were derived from the melanocyte type Myo5a [35] or the brain type Myo5a, which was generated from the melanocyte type Myo5a by inserting exon-B and deleting exons-D and -F as described [36]. The cDNAs of the truncated tail domains of Myo5b and Myo5c were amplified by PCR with the plasmids encoding Myo5b and Myo5c as template [37, 38] and subcloned into the bacterial expression vector pET30HFa (a modified pET30a vector encoding His-tag and Flag-tag). The recombinant tail domains of myosin-5 containing an N-terminal His-tag and Flag-tag were expressed in BL21(DE3) E. coli and purified by Ni-agarose affinity chromatography using standard procedures.

The cDNAs of G3BP1 and G3BP2 were amplified by RT-PCR with the total RNA from fetal mouse brain as the template. The cDNAs encoding full-length G3BP1 and G3BP2 and truncated proteins were subcloned into the pGEX4T2 vector (Amersham Biosciences) using BamHI site and XhoI site. Point mutations were introduced using Fast Mutagenesis System (TransGen) according to the manufacturer’s instructions. The recombinant proteins were expressed in BL21(DE3) E.coli as GST-tagged proteins and purified by GSH-Sepharose affinity chromatography using standard procedures. The His-GST-G3BP1 and its truncated fragments were subcloned into pFastHTc (Invitrogen) from pGEX4T2 by using BamHI and XhoI sites. Recombinant baculovirus was generated using Bac-to-Bac system. The recombinant proteins expressed in Sf9 insect cells was purified by Ni-agarose.

Primary antibody

Anti-Myo5a antibody was generated by immunizing rabbits with the residues 909 to 1234 of mouse Myo5a (Myo5a-909–1234) as follows. The cDNA of Myo5a-909–1234 was amplified by PCR using mouse Myo5a cDNA as the template, cloned into pGEX4T2 vector and pET30a vector, and expressed in BL21(DE3) E.coli as GST-tagged protein (GST-Myo5a-909–1234) and His-tagged protein (His-Myo5a-909–1234), respectively. His-Myo5a-909–1234 was purified using Ni–NTA agarose chromatography. GST-Myo5a-909–1234 was bound to a GSH-Sepharose column, followed by incubation with thrombin for the cleavage of the GST tag, and the eluted Myo5a-909–1234 was used for immunizing rabbits. Anti-Myo5a antibody was affinity-purified from antisera over a column of His-Myo5a-909–1234 coupled to cyanogen bromide-activated Sepharose 4B (GE Healthcare) using standard procedures.

Other primary antibodies for immunofluorescence and Western blot are mouse anti-G3BP1 (PTM BIO, PTM-5700,; 1:200 for immunofluorescence and 1:1000 for Western blot;), and rabbit anti-TIA1 (PTM-5518, PTM BIO; 1:200 for immunofluorescence), rabbit anti-β-tubulin (PTM-6414, PTM BIO; 1:200 for immunofluorescence); Mouse anti-β-actin (CW0096M, cwbio; 1:1000 for Western blot).

Yeast two-hybrid assay

Co-culturing a human kidney cDNA library strain (Y187) with a BrM5a-Tail bait-expressing reporter strain (AH109), the human kidney cDNA fusion library was screened using the BrM5a-Tail/pGBKT7 fusion vector in a yeast-mating approach. A mixture of 100 μl of AH109 strain containing the bait and 100 μl of the human kidney cDNA library strain (Y187) was prepared. To this mixture, 400 μl of YPDA liquid medium was added to the centrifuged pellet, and yeast mating was facilitated at 30 °C with shaking for 8 h. The mating-resultant zygoate cells from mating were spread on QDO to select for putative positive two-hybrid interactions. Yeast single colonies that grew normally after 3–7 days on the medium were selected based on their size (able to grow to > 2 mm). Subsequently, the putative positive colonies were further characterized using QDO/X-α-Gal. The interaction intensity was inferred from the color intensity (shades of blue), and distinctly positive colonies were rescued through plasmid Segregation in bacteria experiments and then identified by sequencing.

GST pulldown assay

GST pulldown assays were performed as described previously [39]. For GST pulldown of GST-G3BP1 or G3BP2 with Flag-BrM5a-Tail, GSH-Sepharose beads (10 μl) were first bound with GST-G3BP1 or G3BP2 by incubating with 95 μl of 2.5 μM GST-tagged protein in Pulldown Buffer (5 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM DTT and 1 mM EGTA) with rotation at 4 °C for 1 h. The GSH-Sepharose beads were washed 3 times with 200 μl of Washing Buffer (10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, and 1 mM EGTA, 1% NP-40) to remove the unbound GST proteins, then mixed with 95 μl of 5 μM Flag-BrM5a-Tail in Pulldown Buffer with rotation at 4 °C for 1 h. Finally, the GSH-Sepharose beads were washed 5 times with 200 μl of Washing Buffer and then eluted by Elution Buffer (10 mM GSH, 50 mM Tris–HCl (pH 8.0), 1 mM DTT, and 200 mM NaCl). The inputs and the eluted proteins were analyzed by SDS-PAGE and visualized by Coomassie Brilliant Blue (CBB) staining or Western blotting using the indicated antibody.

Immunoprecipitation

To detect the interaction between Myo5a and G3BP1 under sodium arsenite treatment, immunoprecipitation was performed following established protocols [40]. Briefly, U2OS cells were cultured in 6-well plates for over 24 h. The cells were then transfected with the Flag-BrM5a-Tail/pcDNA3.1 plasmid. Two days after transfection, the cells were either treated with or without 100 µg/ml cycloheximide for 30 min, followed by exposure to 500 μM sodium arsenite for 1 h or left untreated. Following this, the cells were harvested in lysis buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5% NP40, 100 mM PMSF, 2% protease inhibitor cocktail) and combined with 10 μl of anti-FLAG M2 affinity agarose, then rotated at 4 ℃ for 2 h. Finally, the anti-FLAG M2 affinity agarose was washed 4 times with 200 μl of Washing Buffer (5 mM Tris–HCl, 100 mM NaCl, 1 mM EGTA, 1 mM DTT) and eluted with 0.1 mg/ml FLAG peptide in Washing Buffer. The inputs and the eluted proteins were detected by Western blot using the indicated antibody.

Microscale thermophoresis

The equilibrium dissociation constant (Kd) was measured using the Monolith NT.115 from Nanotemper Technologies. The GST-G3BP1-RBD was fluorescently labeled with ATTO-488 NHS ester (ATTO-TEC GmbH) according to the manufacturer’s protocol. A range of concentrations of ligands (His-tagged McM5a-MTD and McM5a-MTDΔG, ranging from 40 μM to 1.2 nM) was incubated with 20 nM of ATTO-488-labeled GST-G3BP1-RBD in 65 mM NaHCO3, 5 mM Tris–HCl (pH 7.5), 125 mM NaCl, 1 mM DTT, 0.05% Tween 20. The samples were loaded into the Nano Temper glass capillaries and microthermophoresis was carried out using 20% Excitation power and 60% MST power. The Kd values were calculated based on the mass action equation using KaleidaGraph (Synergy Software, Reading, PA). Duplicate reads of triplicate experiments were performed for each interaction.

Identification of Myo5a isoforms expressed in U2OS and A549 cells by RT-PCR

Total RNA was extracted from U2OS and A549 cells using TRIzol (Thermo Fisher), and reverse transcription was performed according to the protocol of the FastKing cDNA first chain synthesis kit (KR116, TIANGEN). Fragment 1 was amplified using a pair primers (F1, 5′-AAGGAAGAAGAAAGGCCACAG-3′; R1, 5′-AGGGCCTCAGCCTCATTCTCA-3′) corresponding to the coding sequence upstream of exon-A and that of exon-E of Myo5a, respectively. Fragment 2 was amplified using a pair primers (F2, 5′-AGAATCCCAGCTACAGTCAC-3′; R2, 5′-TCGATGATCTGTCCTGGAGA-3′) corresponding to the coding sequence of exon-D and that downstream of exon-G of Myo5a, respectively. Fragments 1 and 2 were subjected to DNA sequencing.

Mammalian cells culture and transfection

U2OS cells and A549 cells were provided by Junjie Hu Lab (Institute of Biophysics, Chinese Academy of Sciences) and Xuming Zhou Lab (Institute of Zoology, Chinese Academy of Sciences), respectively, and were cultured as described [41, 42]. Briefly, U2OS cells and A549 cells were cultured in DMEM medium (HyClone) supplemented with 10% fetal bovine serum and penicillin/streptomycin (1% v/v) in a humidified chamber (37 °C, 5% CO2 incubator). Transfections of U2OS cells and A549 cells were performed using Hieff Trans Liposomal Universal Transfection Reagent (Yeasen Biotechnology, Shanghai, China). The plasmids for transfection were created by inserting the cDNA of McM5a-MTD and McM5a-MTDΔG into the pDsRed-N1 vector using XhoI and HindIII sites.

Induce of SG and immunostaining of SG proteins in mammalian cells

Cells were grown in 24-well plates on the coverslips and transfected after 24 h of culturing. Two days after transfection, the cells were treated with 500 μM sodium arsenite in conditioned medium for 1 h. Subsequently, cells were fixed using 4% paraformaldehyde for 15 min, permeabilized with 0.4% Triton X-100 in PBS for 15 min, and then blocked with 1% BSA for 1 h, with all steps performed at room temperature. U2OS cells were incubated with primary antibodies in blocking buffer overnight at 4℃, then washed with PBS for 3 times and incubated with secondary antibodies (anti-mouse IgG coupled with CoraLite 488, Proteintech; anti-rabbit IgG coupled with DyLight 549, Jackson) for 1 h at room temperature. Nuclei were counter-stained with DAPI. The images were captured by Leica STELLARIS 5 fluorescence microscope.

For cycloheximide treatment, U2OS cells were incubated with 100 μg/ml cycloheximide for 30 min at 37 ℃, prior to inducing SGs by sodium arsenite. To examine the effects of myosin inhibitors on SG formation, the cells were pretreated with the indicated myosin inhibitors (PBP or Blebbistatin) in DMEM containing 10% FBS for 1 h at 37 °C, prior to inducing SGs by sodium arsenite as described above. For nocodazole treatment, U2OS cells were incubated with 6.6 μM nocodazole for 2 h at 37 ℃, prior to inducing SGs by sodium arsenite. For nocodazole and PBP treatments, U2OS cells were first exposed to 6.6 μM nocodazole for 1 h, followed by treatment with indicated myosin inhibitors (PBP or Blebbistatin) with 6.6 μM nocodazole in DMEM containing 10% FBS for 1 h at 37 °C, prior to inducing SGs by sodium arsenite.

Generation of Myo5a.−/− U2OS cells by CRISPR-Cas9

Myo5a−/− U2OS cells were generated using CRISPR/Cas9-mediated editing system. The targeting sequences for the Myo5a sgRNAs were as follows: 5′-CACCGGAGCAAGAAGAATATATGA-3′ (targeting exon 12) and 5′- CACCGGATCCAGAAGACCATCCGA-3′(targeting exon 19). The sgRNA expression plasmids were generated by inserting annealed primers into the vector HP180-CBH-Cas9-CMV-EGFP [43]. U2OS cells were transiently transfected with the sgRNA expression plasmids and cultured for 2 days. Single GFP-positive cells were sorted into 96-well plates using a fluorescence-activated cell sorting sorter (BD LSRFortessa SORP). Genomic DNA of from the Myo5a gene of Myo5a−/− cells were amplified by PCR using the following primers (5′-TGTAAATCATGCTTCTCCCGTGAGG-3′ and 5′-GGTCTAGTGTCCCTTTGGAATGAAA-3′ for exon 12; 5′-CTTCTATTTGCTCAACTT-GCATAGG-3′ and 5′-GTTATCTTTTCAGCTATGGTGACCA-3′ for exon 19) and subjected to DNA sequencing. The expression of Myo5a in U2OS cells was examined by Western blot of cell lysate using antibody against mouse Myo5a.

Quantification of the number and size of SGs

The G3BP1 immunofluorescence images were imported into image J. The threshold was adjusted to ensure that all SGs within the field of view were visible. The area of each granule was automatically detected by image J with the granule area of 0.2–25 μm2 and circularity of 0.2–1. All relevant cells were analyzed regardless of presence or absence of SGs. The granules were arranged from smallest to largest and the cumulative areas were calculated. The data were normalized to give the cumulative area of SGs per individual cell. The SG size on the X-axis represents the SGs from all analyzed cells considered individually. The curve of cumulative area of SGs versus SGs size was plotted using GraphPad Prism 9.