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www.rsc.org/ibiology Page 1 of 30 Integrative Biology
It has become widely recognized in the field of mechanobiology that cell behavior is regulated by physical parameters of the niche, including its stiffness. While critical observations have been made regarding the molecular details of this regulation, e.g. translocation of YAP/TAZ, the proteins or complexes that actually convert biophysical to biochemical signals that the cell can interpret remain uncertain. Here we have developed an assay that enables one to predict which proteins could be mechanically sensitive by determining their effect on stem cell differentiation (although other metrics could be substituted). We then identify several focal adhesion mechanosensors and validate them using conventional molecular biology methods.
Manuscript Accepted Biology Integrative Integrative Biology Page 2 of 30
1 High content image analysis of focal adhesion-dependent mechanosensitive stem cell
2 differentiation
3 Andrew W. Holle 1†, Alistair J. McIntyre 1, Jared Kehe 1, Piyumi Wijesekara 1, Jennifer L.
4 Young 1†, Ludovic G. Vincent 1, and Adam J. Engler 1,2*
5
6 1Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA
7 2Sanford Consortium for Regenerative Medicine, La Jolla, CA, USA
8 *Corresponding Author: 9500 Gilman Drive, MC 0695
9 La Jolla, CA 92093 Manuscript
11 858-246-0678
12 †Current Address: Department of New Materials and Biosystems, Max Planck Institute
13 for Intelligent Systems, Stuttgart, Germany Accepted
14
15 Running Title: “Focal adhesion proteins and mechanosensitive differentiation”
16 Keywords: SORBS1, ponsin, stiffness, hydrogel Biology
17
18 Manuscript Information:
19 Abstract: 174 words
20 Body: 6294 words (including references and figure legends) Integrative 21 Figures: 5
22 Supplemental Figures: 5
23 Supplemental Tables: 3
Page 3 of 30 Integrative Biology
24 Abstract
25 Human mesenchymal stem cells (hMSCs) receive differentiation cues from a number of
26 stimuli, including extracellular matrix (ECM) stiffness. The pathways used to sense
27 stiffness and other physical cues are just now being understood and include proteins
28 within focal adhesions. To rapidly advance the pace of discovery for novel
29 mechanosensitive proteins, we employed a combination of in silico and high throughput
30 in vitro methods to analyze 47 different focal adhesion proteins for cryptic kinase binding
31 sites. High content imaging of hMSCs treated with small interfering RNAs for the top 6
32 candidate proteins showed novel effects on both osteogenic and myogenic differentiation; Manuscript
33 Vinculin and SORBS1 were necessary for stiffness-mediated myogenic and osteogenic
34 differentiation, respectively. Both of these proteins bound to MAPK1 (also known as
35 ERK2), suggesting that it plays a context-specific role in mechanosensing for each
36 lineage; validation for these sites was performed. This high throughput system, while Accepted
37 specifically built to analyze stiffness-mediated stem cell differentiation, can be expanded
38 to other physical cues to more broadly assess mechanical signaling and increase the pace
39 of sensor discovery. Biology
40 Integrative
Integrative Biology Page 4 of 30
41 Introduction
42 Although physical properties of the niche have become widely recognized for their
43 influence on a host of cell behaviors 1-3, significant attention has been paid to the
44 influence of extracellular matrix (ECM) stiffness on stem cells 4-6. While initially
45 reported to be myosin contractility sensitive 7, their upstream mechanisms have remained
46 unclear. Recently, however, mechanisms have been proposed involving the nucleus 8,
47 translocation of factors to the nucleus 9, Rho GTPases 10 , stretch activated channels 11 ,
48 and focal adhesions, i.e. “molecular strain gauges” 12 . While numerous mechanisms may
49 overlap, it is clear from these examples that many sensors within each category are still Manuscript
50 undetermined.
51
52 High throughput systems 13 to assess mechano-signaling have yet to play as significant a
53 role as they have in other biomedical and engineering contexts, e.g. biomaterial Accepted
54 microarrays to explore niche conditions 14,15 and microcontact printing to explore the
55 influence of cell shape 16 ; this may be due to fabrication limitations with small volume
56 hydrogels, imaging limitations with thick hydrogels at high magnification, and biological Biology
57 limitations with high throughput molecular screening in stem cells. For example,
58 hydrogels are often fabricated in larger 6- and 24-well formats 7,17,18 and have been used
59 to investigate how a variety of niche properties influence cells 19 . Creating
60 physiologically relevant substrates in small volumes to elicit appropriate cell behaviors is Integrative 61 challenging but not unprecedented 20 ; ensuring that the imaging plane is flat in such small
62 wells, however, has proven difficult and has limited high resolution imaging required for
63 many stem cell applications. Several groups have pursued high throughput imaging of
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64 cells on soft surfaces 21,22 , although these efforts were performed in open culture systems
65 where media interacting with cells on one surface condition was free to diffuse to cells on
66 other surface conditions. Despite these challenges, it is clear that discovery of novel
67 proteins that convert mechanical forces into biochemical signals, e.g. phosphorylation,
68 will require screening due to the sheer number of proteins that could be involved in each
69 mechanism type 8-12 .
70
71 To create a high throughput screen of potential mechanosensing proteins and determine
72 their effects on stem cells, high content screening analysis of multiple cell parameters for Manuscript
73 phenotyping 23,24 is required in addition to high throughput screening systems 25 . While
74 this combination has been used in pre-fabricated small interfering RNA (siRNA) 26 or
75 polymer arrays 15 to examine stem cell pluripotency, their combination in a high
76 throughput array to study mechanically sensitive stem cell differentiation has been Accepted
77 technically challenging. Attempts to leverage high throughput hydrogel systems with
78 high content imaging has been limited by an inability to perform high magnification
79 single cell imaging or investigate the immunofluorescence expression of individual Biology
80 transcription factors 20,27 .
81
82 Here, we have overcome the imaging challenges associated with the 96 well hydrogel
83 array format 20 and combined it with a focal adhesion siRNA screen to determine novel Integrative 84 proteins that convert mechanical forces into biochemical responses, whether acting as
85 direct or indirect transducers of force. We report the identification of several protein hits
86 that may regulate lineage-specific, substrate stiffness dependent differentiation.
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87
88 Experimental
89 Cell Culture and Reagents
90 Human mesenchymal stem cells (Lonza) were maintained in growth medium (DMEM,
91 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin) which was changed every
92 four days (except in 96 well plates). Only low passage hMSCs were used for
93 experimental studies, i.e. less than passage 9. For MAPK1 inhibition, the MAPK1
94 inhibitor pyrazolylpyrrole, dissolved in DMSO, was used at a final concentration of 2 nM
95 and added to cells immediately post-plating. At 2 nM, pyrazolylpyrrole is extremely Manuscript
96 selective and has only been shown to inhibit MAPK1, limiting potential off-target effects
97 28 . Non-differentiation based experiments, including western blots and durotaxis assays,
98 were performed after 24 hours while siRNA-induced protein knockdown was at a
99 maximum. Conversely, differentiation experiments took place over the course of four Accepted
100 days, since differentiation occurs as the integration of cues over time. Cells were plated
101 at a density of 500 cells/cm 2, a sparse density that reduces the likelihood of density-
102 dependent cell signaling over the course of the experiment. Biology
103
104 Polyacrylamide Hydrogel Fabrication in 6- and 96-Well Formats
105 For MAPK1 inhibitor experiments performed in six well plates, acrylamide was
106 polymerized on aminosilanized coverslips. A solution containing the crosslinker N, N’ Integrative 107 methylene-bis-acrylamide, the monomer acrylamide, 1/100 volume 10% Ammonium
108 Persulfate and 1/1000 volume of N, N, N’, N’-Tetramethylethylenediamine was mixed.
109 Two different combinations of acrylamide and bis-acrylamide were used to make
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110 hydrogels of 11 and 34 kilopascal (kPa; a unit of stiffness). Approximately 50 µL of the
111 mixed solution was placed between 25 mm diameter aminosilanized coverslips and a
112 chlorosilanized glass slide for 6-well plates. 100 µg/mL collagen I was chemically
113 crosslinked to the substrates using the photoactivatable crosslinker Sulfo-SANPAH
114 (Pierce). Custom 96 well plates containing collagen type I-conjugated polyacrylamide
115 hydrogels crosslinked to glass bottom surfaces (Matrigen) were fabricated containing
116 equal numbers of 15 kPa wells and 42 kPa hydrogels to induce myogenesis and
117 osteogenesis, respectively (Figure S1A). Stiffness values were verified using an MFP3D-
118 Bio atomic force microscope (Asylum Research, Santa Barbara, CA) using previously Manuscript
119 established methods (Figure S1B) 29,30 . Polyacrylamide gel thickness was also verified
120 using a BD CARV II confocal microscope (Figure S1C,D) and found to be approximately
121 250 µm, which is thick enough that the cells are unable to feel the glass substrate below
122 the gel 31 . Accepted
123
124 siRNA Transfection
125 siRNA oligonucleotides against human Vinculin, p130Cas, SORBS1 (Ponsin), SORBS3 Biology
126 (Vinexin), Palladin, Paxillin, and Filamin (ON-TARGETplus SMARTpool; Thermo
127 Fisher Scientific, Waltham, MA) and a pool of four non-targeting siRNAs control
128 oligonucleotides (ON-TARGETplus siControl; Dharmacon), diluted in DEPC water
129 (OmniPure, EMD) and 5X siRNA buffer (Thermo Fisher Scientific, Waltham, MA), Integrative 130 were transiently transfected into human hMSCs using Dharmafect 1 (Thermo Fisher
131 Scientific, Waltham, MA) at an optimized concentration of 50 nM in low serum
132 antibiotic free growth media, according to the manufacturers’ protocols. Specific siRNA
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133 sequences can be found in Supplemental Table 1. Protein knockdown was characterized
134 by western blot and immunofluorescence. After 24 hours of transfection in antibiotic-
135 free media (2% FBS), media was replaced with standard hMSC growth media and cells
136 replated onto appropriate substrates.
137
138 Plasmid Transfection
139 pEGFP-C1 sub-cloned with complete Vinculin cDNA, which had been originally excised
140 from p1005 with EcoRI and inserted in EcoRI digested pEGFP-C1 (labeled as FL), was
141 obtained from Dr. Susan Craig 32 . L765I mutant Vinculin plasmids were obtained via Manuscript
142 site-directed mutagenesis on FL Vinculin plasmids. All plasmids were purified using
143 QIAGEN Plasmid Midi Kit (Qiagen). hMSCs were transfected in antibiotic-free medium
144 with 1 mg of plasmid precomplexed with 2 µl of Lipofectamine 2000 (Life Technologies)
145 in 100 µl of DMEM. After 24 hours of transfection in antibiotic-free media with 2% FBS, Accepted
146 media was replaced with standard hMSC growth media.
147
148 Immunofluorescence Biology
149 hMSCs were fixed with 3.7% formaldehyde for 30 minutes at 4 oC and permeabilized
150 with 1% Triton-X for 5 minutes at 37 oC. The cells were then stained with primary
151 antibodies against human MyoD (sc-32758, Santa Cruz), Myf5 (sc-302, Santa Cruz,
152 Dallas, TX), Osterix (ab22552, Abcam), CBFA1 (RUNX2) (sc-101145, Santa Cruz), Integrative 153 Vinculin (ab129002, Abcam), p130Cas (ab108320, Abcam), SORBS1 (ab4551, Abcam),
154 SORBS3 (GTX-115362, Genetex), Filamin (ab51217, Abcam), or Paxillin (ab32084,
155 Abcam). Corresponding secondary antibodies were conjugated to Alexa Fluor 488
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156 (FITC) or Alexa Fluor 647 (Cy5) (Invitrogen). Nuclei were counterstained with Hoechst
157 dye (Sigma), and the actin cytoskeleton was stained with rhodamine-conjugated
158 phalloidin (Invitrogen). Cells not plated in 96 well plates were imaged with a Nikon
159 Eclipse Ti-S inverted fluorescence microscope equipped with a BD Carv II camera.
160
161 High Content Imaging and Analysis
162 96 well plates were imaged on a CV1000 Cell Voyager (Yokogawa). Briefly, images
163 were acquired through 5 z-positions with 10 µm step sizes at 25 different points in each
164 well with three different filter sets (FITC, TXRD, and DAPI). Maximum Intensity Manuscript
165 Projections (MIPs) were constructed from the resulting stitched z-stacks to account for
166 uneven, slanted, or differentially swollen hydrogel surfaces and analyzed using a semi-
167 automated image analysis pipeline in CellProfiler 33 . Nuclear outlines were obtained as
168 primary objects with automatic Otsu Global thresholding (Figure S2A) and cell outlines Accepted
169 were obtained using the TXRD channel as secondary objects using a Watershed Gradient
170 algorithm (Figure S2B). The pipeline calculated morphological attributes (such as cell
171 area, aspect ratio, and eccentricity) for each cell, as well as the mean and integrated Biology
172 density of the FITC channel signal in nuclei, cell outlines, and cytoplasm outlines. From
173 these data, one could distinguish cells with nuclear expression only, cytoplasm
174 expression only, uniform positive expression, and uniform negative expression, as shown
175 with example cells in Figure S2C. Data analysis was performed with Microsoft Excel, Integrative 176 GraphPad Prism, and CellAnalyst 34 .
177
178 Western Blots
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179 Cell lysates were collected by rinsing samples with cold PBS, followed by a five minute
180 lysis in mRIPA buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1%
181 Triton, 1% Na-DOC, 0.1% SDS) with 1 mM EGTA, 1 mM Na3VO4, 10 mM Na4P2O7,
182 and 1 mM PMSF (protease inhibitors). Cell lysates were separated via SDS-PAGE,
183 transferred to PVDF membranes (Bio-Rad), and washed in Buffer A (25 mM Tris-HCl,
184 150 mM NaCl, 0.1% Tween-20) + 4% SeaBlock (Thermo Fisher Scientific, Waltham,
185 MA) overnight at 4 oC. Membranes were incubated with anti-Vinculin (ab18058,
186 Abcam), GAPDH (ab8245, Abcam), ERK2 (ab7948, Abcam), p130Cas (ab108320,
187 Abcam), SORBS1 (ab4551, Abcam), SORBS3 (GTX-115362, Genetex), Filamin Manuscript
188 (ab51217, Abcam), or Paxillin (ab32084, Abcam) antibodies for 1 hour, washed with
189 Buffer A containing SeaBlock, and incubated in streptavidin horseradish-peroxidase-
190 conjugated secondary antibodies (Bio-Rad) for 30 minutes at room temperature.
191 Immunoblots were visualized using ECL reagent (Pierce). All western blot antibodies Accepted
192 were obtained from Abcam (Cambridge, England).
193
194 Quantitative PCR Biology
195 mRNA was isolated from hMSCs grown after 4 days with Trizol, and subsequently
196 treated with chloroform and precipitated with isopropanol. The cell lysate was
197 centrifuged and the pellet washed in ethanol twice, after which the pellet was allowed to
198 dry before resuspension in DEPC water. cDNA was assembled through reverse Integrative 199 transcriptase polymerase chain reaction (RT-PCR) for one hour at 37 oC, followed by a 5
200 minute inactivation step at 99 oC. 1 µL of the resulting cDNA mixture was added to 12.5
201 µL SYBR Green Real Time PCR Master Mix (Thermo Fisher Scientific, Waltham, MA)
Page 11 of 30 Integrative Biology
202 containing 0.25 nM forward and reverse primers (Supplemental Table 2) and enough
203 DEPC water to bring the total reaction volume per well to 25 µL.
204
205 Immunoprecipitation
206 Cell lysates were collected with a non-denaturing lysis buffer (20 mM Tris-HCl pH 8,
207 127 mM NaCl, 1% Nonidet P-40, 2 mM EDTA). Anti-ERK2 antibody (Abcam
208 ab124362) was bound to protein G-conjugated Dynabeads (Life Technologies, Carlsbad,
209 CA, USA) for 1 hour at 4 oC with gentle agitation. Beads were magnetically captured, the
210 supernatant removed, and the pellet incubated overnight at 4 oC before Western Blot Manuscript
211 analysis.
212
213 Statistics
214 All experiments were performed in triplicate with the indicated number of cells analyzed Accepted
215 per condition. Error bars shown are the standard error of the mean (SEM). Significance
216 was assessed with Bonferroni’s Multiple Comparison Test at a significance threshold of
217 p<0.05 or lower as indicated. Values less than 0.1 were noted. For instances where data is Biology
218 not significantly different, N.S. is stated.
219
220 Results
221 Bioinformatic Assessment of Focal Adhesion-based Mechanosensing Reveals that Integrative 222 MAPK1 Binding is Frequent and Cryptic
223 We selected 47 focal adhesion proteins 35,36 (Supplemental Table 3) based on their ability
224 to bind multiple proteins at their N- and C-terminal ends such that they could potentially
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225 be unfolded when one end of the protein is displaced relative to the other, i.e. a
226 “molecular strain sensor” 12 . These candidates were analyzed with ScanSite 37 , a tool
227 designed to identify short protein sequence binding motifs and predict whether the motif
228 is surface accessible. After analyzing all 47 proteins, a scatter plot showing the number
229 of times a predicted binding site was found versus the average accessibility of the
230 identified sites was constructed (Figure 1A, Supplemental Table 3). Interestingly,
231 predicted MAPK1 binding sites were found most frequently and with the second-lowest
232 average accessibility, implying that MAPK1 is the most likely candidate to affect stem
233 cell differentiation across a wide variety of cellular pathways in a manner that requires a Manuscript
234 change in surface accessibility of the MAPK1 binding site.
235
236 MAPK1 Inhibition Prevents Mechanosensitive Stem Cell Myogenesis and Osteogenesis
237 To analyze the effect of MAPK1 inhibition on substrate stiffness-directed hMSC Accepted
238 differentiation, MAPK1 was inhibited with pyrazolylpyrrole, an extremely potent and
239 selective MAPK1 inhibitor 38 , immediately post plating in order to limit any early
39
240 mechanosensing events, which can occur on the time scale of minutes . Consistent with Biology
241 previous results 17 , we found that hMSCs exhibited a 50% reduction in nuclear-localized
242 myogenic transcription factors MyoD and Myf5 after 4 days in culture (Figure 1B). On
243 34 kPa substrates, pyrazolylpyrrole-treated hMSCs also exhibited reduced osteogenic
244 transcription factor expression and localization (Figure 1B). However, since Integrative 245 pyrazolylpyrrole is a global MAPK1 inhibitor, it may inadvertently reduce lineage
246 commitment through non-stiffness mediated mechanisms.
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247 To address this, the potentially upstream focal adhesion proteins identified by ScanSite
248 were investigated further. Of the proteins analyzed, five–Vinculin, p130Cas, Filamin,
249 SORBS1 (Ponsin), SORBS3 (Vinexin)–had a predicted cryptic MAPK1 binding site and
250 terminal multiple binding sites to other proteins, which would allow the protein to be
251 strained and change configuration under an appropriate amount of force, F* (Figure 1C).
252 This conformation change could then expose the MAPK1 binding site predicted to be
253 cryptic, but only under the appropriate amount of force. Paxillin was selected as a
254 control protein because it did not have a cryptic MAPK1 binding site (Figure S3).
255 siRNAs were used to transiently knock down candidate proteins, which was verified by Manuscript
256 western blot (Figure 2A) and immunofluorescence (Figure 2B-C). siRNA-induced
257 knockdown of these proteins did not affect endogenous expression of MAPK1 (Figure
258 S5).
259 Accepted
260 To analyze whether siRNA-induced knockdown of the five candidate proteins could alter
261 mechanically-sensitive myogenic and osteogenic differentiation, hMSCs were cultured in
262 96 well plates containing polyacrylamide hydrogels of roughly 250 µm thickness and Biology
263 stiffness of either 15 kPa (myogenic) or 42 kPa (osteogenic) for four days (Figure S1).
264 These stiffness values are within the characteristic ranges of myogenic- and osteogenic-
265 inducing 2D substrates 7,40-42 . To analyze osteogenesis, cells in the 42 kPa wells were
266 fixed and stained for the osteogenic transcription factors Osterix and CBFA1, while for Integrative 267 myogenesis, cells in the 15 kPa wells were fixed and stained for the myogenic
268 transcription factors MyoD and Myf5. Expression levels for the transcription factors
269 were compared with those in untreated cells at day 0 (negative control) and at day 4
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270 (positive control) on the corresponding stiffness hydrogels. Transcription factors were
271 specifically chosen as outputs for identifying mechanosensitivity because both the
272 expression and nuclear localization could be used as criteria for lineage commitment
273 (Figure S2). To further reduce the false discovery rate, we only classified a protein as a
274 mechanosensor if their knockdown impaired stiffness-induced differentiation as assessed
275 by both transcription factors. As transcription factor expression is often sequential, this
276 reduces the likelihood that the assay simply missed the time when the transcription factor
277 was active.
278 Manuscript
279 In the osteogenesis assay, we found that p130Cas, Filamin, Paxillin, and SORBS3
280 (Vinexin) knockdown did not affect osteogenic differentiation signals after 4 days
281 relative to day 0 expression and localization. Conversely, the knockdown of SORBS1
282 (Ponsin), which interacts with Vinculin 43 and plays a role in insulin signaling 44 , reduced Accepted
283 both CBFA1 and Osterix nuclear expression by over 50%. Vinculin knockdown, which
284 was previously shown to not affect CBFA1 expression 17 , slightly reduced CBFA1 but
285 not Osterix expression (Figure 3); no myogenic expression was found in these cells (data Biology
286 not shown). Thus, we concluded that SORBS1 could act as a unique stiffness-mediated
287 sensor for osteogenic differentiation.
288
289 In the myogenesis assay, siRNA knockdown of Vinculin, p130Cas, or SORBS3 resulted Integrative 290 in a loss of stiffness-induced expression of both MyoD and Myf5 at day 4. This is in
291 agreement with recent reports of Vinculin-mediated SORBS3 mechanosensing 45 .
292 However, Filamin, SORBS1, and Paxillin only reduced expression of one of the two
Page 15 of 30 Integrative Biology
293 myogenic markers (Figure 4). Paxillin does not contain a cryptic MAPK1 binding site,
294 so Myf5 reduction may be due to other predicted cryptic binding domains that it contains,
295 e.g. MAPK3; no osteogenic expression was found in these cells (data not shown). Thus,
296 we concluded that Vinculin could act as a unique stiffness-mediated sensor for myogenic
297 differentiation, consistent with prior reports 17 .
298
299 If knockdown of the candidate focal adhesion proteins disrupts not just mechanosensitive
300 signaling but also other normal cell behaviors, stiffness-mediated differentiation
301 differences may not solely be related to signaling. High content image analysis was Manuscript
302 performed with CellProfiler to measure cell area and morphology, i.e. eccentricity, of
303 cells from all conditions. Neither area nor morphology was altered by any of the siRNA
304 treatments (Figure S4A-B). Cell migration speed was also unaffected by siRNA
305 knockdown, although SORBS3 knockdown appeared to increase migration persistence Accepted
306 (Figure S4C). Perhaps most importantly, focal adhesion assembly in terms of size and
307 distribution appeared unaffected in single knockdown experiments; outside of the
308 expected loss of expression of the proteins being knocked down, no changes were Biology
309 observed in these focal adhesion characteristics (Figure S4D). Differentiation changes
310 could also be due to off-target effects of the siRNA on MAPK1 expression, thus
311 depleting the endogenous pool of the sensor’s binding partner and inadvertently
312 preventing differentiation. However, MAPK1 western blots indicated that knockdown did Integrative 313 not impact endogenous expression (Figure S5), reinforcing the concept that individual
314 mechanosensing proteins regulated transcription factor expression.
315
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316 Validation of Candidate Mechanosensor Hits for MAPK1 Interaction
317 To verify hits directly using more targeted molecular methods, SORBS1 was
318 immunoprecipated via MAPK1. For hMSCs cultured for 24 hours on 34 kPa PA gels,
319 SORBS1 was detected in the pellet but not the unconcentrated whole cell lysate,
320 suggesting that, although expressed at low levels, SORBS1 and MAPK1 interact in cells
321 cultured on physiological-stiffness gels (Figure 5A). SORBS1 contains two predicted
322 binding sites for MAPK1 at L500 and L1033 (Figure S2B), but among the twelve
323 SORBS1 isoforms, only two contain the predicted L1033 binding site 46-48 . qPCR
324 indicated that undifferentiated cells cultured on 34 kPa substrates for 24 hours did not Manuscript
325 significantly express SORBS1 isoforms containing L1033 (Figure 5B). Lacking other
326 kinase binding domains predicted with high confidence to be inaccessible (i.e. Scansite
327 accessibility prediction less than 0.5), the MAPK1 binding site found on SORBS1 at
328 L500 is the most likely candidate to act as a stretch sensitive mechanosensor. For Accepted
329 Vinculin, which pulls MAPK1 down with immunoprecipitation on 11 kPa substrates 17 ,
330 MAPK1 binding was predicted at L765 (Figure S2B). To confirm that L765 is
331 specifically required for myogenic differentiation on 11 kPa substrates, a plasmid Biology
332 containing L765I-mutated Vinculin and Green Fluorescent Protein (GFP) was added back
333 to cells that had been treated with Vinculin siRNA. While Vinculin knockdown was
334 sufficient to reduce myogenic transcription factor expression in hMSCs, addback of full-
335 length Vinculin rescued expression whereas addback of L765I-mutated Vinculin was Integrative 336 insufficient to fully rescue expression (Figure 5C, filled vs. open arrowhead,
337 respectively).
338
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339 Discussion
340 While these data specifically focus on screening 47 focal adhesion proteins with a
341 “molecular strain sensor”-like structure as predicted by ScanSite, some of which have
342 never been identified as mechanically sensitive, the list of proteins comprising focal
343 adhesions is much larger and dynamic. Current estimates implicate as many as 232
344 different components, of which 148 are intrinsic and 84 are transient 49 , as a common
345 signature of adhesions. Recent analyses of focal adhesions have even identified more
346 than 1300 distinct proteins within isolated adhesion complexes 50 , suggesting exceedingly
347 complex adhesion-based mechanisms for cells that must actively sense their Manuscript
348 surroundings. Focal adhesion composition and structure have also recently been shown
349 to be relatively stable to external perturbation, including siRNA knockdown or chemical
350 inhibition of components, suggesting that signaling transduction occurs independently of
351 structural integrity 51 . That said, our data also focused on proteins with relatively little Accepted
352 functional data, e.g. SORBS1, to establish proof-of-principle that we can use a high
353 content imaging based platform to identify candidate sensors via their influence on stem
354 cell differentiation. Biology
355
356 Prior to this work, SORBS1, also known as Ponsin, Sorbin, CAP, or c-Cbl associated
357 protein, has not been implicated in mechanosensitive differentiation, although it has been
358 shown to affect actin cytoskeleton organization via Dynamin GTPases 52 , bind to vinculin Integrative 359 43 , and be overexpressed and phosphorylated in response to endogenous PYK2
360 expression, a focal adhesion complex-localized kinase capable of suppressing
361 osteogenesis 53 .
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362
363 Even with a fairly well studied focal adhesion protein like Vinculin, questions about its
364 force-sensitive behavior remain. Vinculin undergoes a conformational change from its
365 autoinhibited state to an ‘activated’ state in which it can bind F-actin, allowing it to
366 transmit force from the cytoskeleton 54 . Studies have shown that vinculin is under
367 mechanical tension within focal adhesions, although the activating conformational
368 change is separable from the application of force across the protein 55 . Recent work has
369 revealed that this tension is independent of substrate stiffness 56 , suggesting that vinculin’s
370 upstream binding partner talin may bear the brunt of force sensing. Intriguingly, talin’s Manuscript
371 unfolding under force is sufficient to expose differential amounts of cryptic vinculin
372 binding sites 57 , meaning that differential amounts of (potentially force-sensitive) vinculin
373 activation can initiate different differentiation pathways. Thus, it is possible that the
374 exposure of the cryptic MAPK1 domain in vinculin occurs after activation, and after talin Accepted
375 and actin binding, in a force dependent manner. While adding a Talin knockdown to our
376 screen would serve as an effective positive control, attempts at siRNA-induced talin
377 knockdown have led to a loss in normal cell morphology (data not shown), likely because Biology
378 of the key structural role it plays in linking the cytoskeleton to focal adhesions.
379
380 Beyond stem cell differentiation assays, several alternative high throughput techniques
381 have been adapted for mechanobiology sensor identification 58 though they do not Integrative 382 utilized biomimetic substrates. For example, mass spectroscopy “cysteine shotgun”
383 assays use cysteine-binding dyes to assess differential protein labeling under stress 59 but
384 this approach focuses on the conformational change itself and may overlook downstream
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385 signaling changes. Even when applied directly to differential unfolding in response to
386 mechanical signals 60 , one could miss transient protein unfolding during signal
387 transduction, especially if cryptic binding domains do not contain cysteine residues.
388 While this RNAi screening approach is more targeted, it can be adapted to fit any
389 instance in which immunofluorescence is used to measure an output, e.g. a response to
390 change in substrate stiffness, and can be specific for nuclear or cytoplasmic expression
391 (Figure S2).
392
393 Conclusions Manuscript
394 A computational approach was used to select candidate proteins that could potentially
395 play a role in MAPK1-based mechanosensitive differentiation based on an analysis of
396 their binding partners and presence of cryptic signaling sites, i.e. the “molecular strain
397 gauge” structure 12 . A high throughput, high content analysis based system capable of Accepted
398 finding hits much more quickly and efficiently was then constructed to test these
399 candidates, with which we identified SORBS1 and Vinculin as potential mechanosensors
400 in hMSCs. While this method was applied specifically to the mechanical influence of Biology
401 stiffness on stem cells differentiation, it can be applied to a number of applications in cell
402 biology in which an immunofluorescently-labeled marker is differentially up- or down-
403 regulated in response to a physical stimulus, e.g. stiffness, etc.
404 Integrative 405 Acknowledgements
406 The authors would like to acknowledge Dr. Jamie Kasuboski for assistance with high
407 content imaging and Dr. Brenton Hoffman for helpful comments on the manuscript. This
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408 work was supported by grants from the National Institutes of Health (DP2OD006460 to
409 A.J.E.) and National Science Foundation (1463689 to A.J.E) as well as a National
410 Science Foundation predoctoral fellowship (to L.G.V.)
411
412 References
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532 PNAS , 2011, 108 , 8269–8274. Integrative 533
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534 Figures:
535 Figure 1: ScanSite Results for 47 Different Focal Adhesion Proteins. (A) Each data
536 point represents a predicted binding partner. The y-axis displays the number of times this
537 binding partner was identified during the analysis of the 47 focal adhesion proteins, while
538 the x-axis shows the average accessibility of the binding site. Predicted surface
539 inaccessible binding sites have accessibility values below 1 (gray region). (B) MAPK1
540 inhibitor pyrazolylpyrrole (MAPKi) was applied to cells at the beginning of the 4-day
541 time course on both (A) 11 kPA and (B) 34 kPa substrates and stained for (A) MyoD
542 (white) or Myf5 (gray) and (B) CBFA1 (white barred) or Osterix (gray barred) as Manuscript
543 indicated on day 4. Mean nuclear fluorescence is plotted normalized to untreated cells.
544 **p<0.01 and ***p<0.001 relative to untreated cells stained for the same transcription
545 factor. (C) Schematic of force-induced conformational changes by a “molecular strain
546 sensor” where proteins bound to the sensor stretch the it by transmitting a force across the Accepted
547 protein. The resulting conformational change exposes the once cryptic binding site at an
548 optimal force, F* (middle schematic). Above or below that value results in excessive
549 deformation of the binding site to prevent binding or not enough stretch causing the site Biology
550 to remain cryptic, respectively.
551
552 Figure 2: Confirmation of siRNA-induced Knockdown. (A) Western blots of lysates
553 collected 2 days post siRNA treatment. (B) Immunofluorescence images of proteins Integrative 554 being knocked down. (C) Quantification of mean immunofluorescence intensity from
555 knockdown cells. For Vinculin, p130Cas, SORBS3, SORBS1, Filamin, and Paxillin in
556 (C), n > 10 cells in triplicate.
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557
558 Figure 3: Osteogenic Differentiation and Focal Adhesion Protein Knockdown.
559 Normalized mean intensity levels of (A) CBFA1 and (B) Osterix immunofluorescence
560 staining after four days of culture of osteogenically favorable 42 kPa substrates.
561 Representative images show cell outlines along with (C) CBFA1 and (D) Osterix
562 expression. Filled arrowheads indicate nuclei that maintained transcription factor
563 expression whereas open arrowheads indicate nuclei that lost expression. (E) Heat map
564 indicating fold-change in expression of the indicated osteogenic markers from day 0 wild
565 type cells. For WT, Vinculin, p130Cas, Filamin, SORBS3, Paxillin, SORBS1, and d0 Manuscript
566 WT in (A) and (B), n=298, 35, 28, 44, 29, 28, 20, and 40, respectively.
567
568 Figure 4: Myogenic Differentiation and Focal Adhesion Protein Knockdown.
569 Normalized mean intensity levels of (A) Myf5 and (B) MyoD immunofluorescence Accepted
570 staining after four days of culture of myogenically favorable 15 kPa substrates.
571 Representative images show cell outlines along with (C) Myf5 and (D) MyoD
572 expression. Filled arrowheads indicate nuclei that maintained transcription factor Biology
573 expression whereas open arrowheads indicate nuclei that lost expression. (E) Heat map
574 indicating fold-change in expression of the indicated myogenic markers from day 0 wild
575 type cells. For WT, Vinculin, p130Cas, Filamin, SORBS3, Paxillin, SORBS1, and d0
576 WT in (A) and (B), n=39, 31, 43, 24, 30, 35, 29, and 9, respectively. Integrative 577
578 Figure 5: Molecular validation of Mechanosensitive Protein Interactions. (A)
579 SORBS1 blots of lysates without (top) or with immunoprecipitation (middle and bottom)
Page 25 of 30 Integrative Biology
580 via a MAPK1 antibody. Supernatant and pellet fractions of the immunoprecipitation are
581 shown (middle and bottom, respectively). Prior to lysis, cells were cultured on 34 kPa
582 substrates. (B) qPCR of SORBS1 using primers that target a conserved portion of the
583 gene (labeled All SORBS1) versus a region only found in the two full length isoforms
584 (labeled L1033). Data is normalized to the GAPDH and then the All SORBS1 condition.
585 Input RNA was collected from hMSCs on 34 kPa substrates for 24 hours. (C) Add back
586 of Full-Length (FL) or mutated Vinculin plasmid (L765I) to Vinculin siRNA-treated cells
587 showing GFP and MyoD expression after 4 days on 11 kPa substrates. Filled and open
588 arrowheads indicate where nuclear localized MyoD expression is or should be. Manuscript Accepted Biology Integrative
Figure 1 Integrative Biology Page 26 of 30
Surface Surface A Inaccessible Accessible 50
40
t MAPK1 n 30 u o
C 20
10
0 0 1 2 3 4 5 B Average Accessibility 1.5 MyoD Myf5 CBFA1 Osterix n o ss i Manuscript e r
p 1.0 x
E *** *** ** 0.5 Normalized
0.0 Accepted Untreated MAPKi Untreated MAPKi Myogenic (11 kPa) Osteogenic (34 kPa) C Exposed, functional binding site Biology
f < F* f = F* f > F* Molecular Strain Sensor MAPK1 Integrative FigurePage 27 of 302 Integrative Biology B WT siRNA WT siRNA A WT siRNA Vinculin Vinculin SORBS1 Filamin
Paxillin Filamin p130Cas p130Cas Manuscript
SORBS1 Paxillin SORBS3 Accepted Vinculin SORBS3 C 1.5 p130Cas *** ** * y
t 1.0 Biology i en s t 0.5
0.0 sc en t I n SORBS1 Filamin Paxillin Integrative
e 1.5 * **** *** uo r l
F 1.0
0.5
0.0 WT KD WT KD WT KD Figure 3 Integrative Biology Page 28 of 30 *** A 1.5 * B * y y t t i i 1.0 en s en s t t
1.0 n I x i r e t s 0.5 0.5 ean O ean CBFA1 I n M M Manuscript 0.0 0.0
T illin T T illin W x W x ilamin a a 0 WT F P P Vinculin d0 W Vinculin Filamin D p130Cas SORBS3 SORBS1 p130Cas SORBS3 SORBS1
C D Accepted Biology
WT Vinculin p130Cas SORBS1 WT Vinculin p130Cas SORBS1 Integrative
Filamin SORBS3 Paxillin Filamin SORBS3 Paxillin
E Osterix CBFA1 1 2 3 Fold Expression from day 0 WT inculin Paxillin Filamin V p130Cas SORBS3 SORBS1 FigurePage 29 of 304 Integrative Biology * ** A 1.5 *** B1.5 *** n n o o ss i ss i e e r r
1.0 p p 1.0 x x E E
5 D My f
0.5 My o 0.5 ean ean M M Manuscript 0.0 0.0 T illin T W x W Vin xillin ilamin a p130 a F P ilamin Vinculin D0 WT F P D0 WT p130Cas SORBS3 SORBS1 SORBS3 SORBS1 C D Accepted Biology WT Vinculin p130Cas SORBS1 WT Vinculin p130Cas SORBS1 Integrative Filamin SORBS3 Paxillin Filamin SORBS3 Paxillin E Myf5 MyoD 2 3 4 Fold Expression from day 0 WT inculin Paxillin Filamin V p130Cas SORBS1 SORBS3 Figure 5 Integrative Biology Page 30 of 30 GFP MyoD A IP: MAPK1 C Lysate B 1.0 Manuscript Vinc FL Accepted
Sup. 0.5 Biology Integrative Blot: SORBS1
Pellet siRNA Vinculin 0.0
All SORBS1 Vinc L765I Normalized Expression L1033