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SHORT REPORT SPECIAL ISSUE: CELL BIOLOGY OF MOTORS
Myosin-X recruits lamellipodin to filopodia tips
Ana Popovic
1,2
, Mitro Miihkinen
1,
*, Sujan Ghimire
1,2
, Rafael Saup
1
, Max L. B. Gro
nloh
1,‡
, Neil J. Ball
3
,
Benjamin T. Goult
3
, Johanna Ivaska
1,4,5, 6,§
and Guillaume Jacquemet
1,2,5, 8,§
ABSTRACT
Myosin-X (MYO10), a molecular motor localizing to filopodia, is
thought to transport various cargo to filopodia tips, modulating
filopodia function. However, only a few MYO10 cargoes have been
described. Here, using GFP-Trap and BioID approaches combined
with mass spectrometry, we identified lamellipodin (RAPH1) as a
novel MYO10 cargo. We report that the FERM domain of MYO10 is
required for RAPH1 localization and accumulation at filopodia tips.
Previous studies have mapped the RAPH1 interaction domain for
adhesome components to its talin-binding and Ras-association
domains. Surprisingly, we find that the RAPH1 MYO10-binding site
is not within these domains. Instead, it comprises a conserved helix
located just after the RAPH1 pleckstrin homology domain with
previously unknown functions. Functionally, RAPH1 supports
MYO10 filopodia formation and stability but is not required to activate
integrins at filopodia tips. Taken together, our data indicate a feed-
forward mechanism whereby MYO10 filopodia are positively regulated
by MYO10-mediated transport of RAPH1 to the filopodium tip.
KEY WORDS: Filopodia, MYO10, RAPH1, Cargo transport, Molecular
motor
INTRODUCTION
Cell migration is essential during embryonic development, immune
surveillance and wound healing. Misregulation of cell migration is
implicated in multiple diseases, including inflammation and cancer.
One hallmark of cell motility is a high degree of plasticity, allowing
cells to adopt different morphologies and migration modes (Conway
and Jacquemet, 2019). A shared feature of efficient cell migration is
the ability of cells to probe and interact dynamically with their
environments using cellular protrusions, such as filopodia,
lamellipodia or pseudopods.
Filopodia are small and dynamic finger-like actin-rich
protrusions (1–5 µm in length and 50–200 nm in width) and are
often the first point of contact between a cell and its immediate
surroundings. Filopodia contain cell surface receptors, such as
integrins, cadherins and growth factor receptors, interacting with
and interpreting various extracellular cues. Filopodia assembly is
primarily driven by the linear polymerization of actin filaments with
their barbed ends facing the plasma membrane (Jacquemet et al.,
2015). These filaments are further organized into tightly packed
bundles by actin-bundling proteins. This unidirectional organization
allows molecular motors, such as myosin-X (MYO10), to walk
along filopodia and accumulate at their tips (at ∼600 nm/s) (Kerber
et al., 2009). By doing so, MYO10 is thought to transport various
proteins to filopodia tips, modulating filopodia function (Jacquemet
et al., 2015; Arjonen et al., 2014; Berg and Cheney, 2002; Hirano
et al., 2011; Zhang et al., 2004). However, only a very few MYO10
cargoes have been proposed to date, with the netrin DCC receptor
(Zhu et al., 2007; Wei et al., 2011), integrins (Zhang et al., 2004;
Wei et al., 2011) and VASP (Tokuo and Ikebe, 2004) being the
principal ones. The MYO10 FERM (protein 4.1R, ezrin, radixin,
moesin) domain has been described as the main cargo-binding site
in MYO10 (Wei et al., 2011). We previously reported that MYO10
FERM was not required to localize integrins or VASP at filopodia
tips. Instead, we found that MYO10 FERM is required for proper
integrin activation at filopodia tips (Miihkinen et al., 2021). In
addition, we found that deleting the MYO10 FERM domain had
little impact on the localization of significant filopodia tip complex
components (Miihkinen et al., 2021). These results lead us to
question the role of MYO10 as a cargo-transporting molecule.
Here, we set out to identify novel MYO10 cargo molecules.
Using GFP-Trap and BioID approaches combined with mass
spectrometry, we identified lamellipodin (RAPH1) as a novel
MYO10-binding partner. Using structured illumination
microscopy, we report that the FERM domain of MYO10 is
required for RAPH1 localization and accumulation at filopodia tips;
thus, RAPH1 is likely an MYO10 cargo. We map the RAPH1
MYO10-binding site to a previously uninvestigated RAPH1
sequence, and demonstrate that RAPH1 is a critical positive
regulator of filopodia formation and stability in cells. Our results
indicate that, in filopodia, RAPH1 is not required for integrin
activation. Instead, RAPH1 regulates MYO10 filopodia formation
and stability.
RESULTS AND DISCUSSION
RAPH1 is a putative MYO10 cargo
To identify novel MYO10 cargo, we searched for proteins that
interact specifically with the MyTH4/FERM domain (termed
MYO10-FERM here for simplicity), the main cargo-binding site
in MYO10 (Wei et al., 2011). We performed GFP pulldowns in
U2-OS cells stably expressing GFP, GFP–MYO10
FERM
,or
Handling Editor: Anne Straube
Received 25 August 2022; Accepted 12 January 2023
1
Turku Bioscience Centre, University of Turku and Åbo Akademi University, 20520
Turku, Finland.
2
Faculty of Science and Engineering, Cell Biology, Åbo Akademi
University, 20520 Turku, Finland.
3
School of Biosciences, University of Kent,
Canterbury, Kent CT2 7NJ, UK.
4
Department of Life Technologies, University of
Turku, 20520 Turku, Finland.
5
InFLAMES Research Flagship Center, University of
Turku and Åbo Akademi University, 20520 Turku, Finland.
6
Western Finnish Cancer
Center (FICAN West), University of Turku, 20520 Turku, Finland.
7
Foundation for the
Finnish Cancer Institute, Tukholmankatu 8, 00014 Helsinki, Finland.
8
Turku
Bioimaging, University of Turku and Åbo Akademi University, 20520 Turku, Finland.
*Present address: Institute for Molecular Medicine Finland (FIMM), Tukholmankatu
8, 00270 Helsinki, Finland.
‡
Present address: Molecular Cell Biology Lab,
Department of Molecular Cellular Hemostasis, Sanquin Research and Landsteiner
Laboratory, 1066 CX Amsterdam, the Netherlands.
§
Authors for correspondence (guillaume.jacquemet@abo.fi;
johanna.ivaska@utu.fi)
B.T.G., 0000-0002-3438-2807; J.I., 0000-0002-6295-6556; G.J., 0000-0002-
9286-920X
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
1
© 2023. Published by The Company of Biologists Ltd
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Journal of Cell Science (2023) 136, jcs260574. doi:10.1242/jcs.260574
Journal of Cell Science
GFP–TLN1
FERM
(the talin-1 FERM domain), followed by mass
spectrometry analysis (Fig. 1A,B). TLN1 FERM was selected as an
additional control as it shares structuralsimilarities with the MYO10
FERM domain but performs different functions in cells (Miihkinen
et al., 2021). We identified 87 proteins that were specifically
enriched in the MYO10-FERM pulldowns (Fig. 1A,B; Table S1).
Interestingly, small GTPase regulators, such as RASAL2,
ARHGDIA and TRIO, were among the enriched putative
MYO10-FERM binders (Table S1).
Next, to narrow the list of putative MYO10 cargo, we tagged
GFP–MYO10 with the promiscuous biotin ligase BioID (Roux
et al., 2012). BioID was tagged in the C-terminal region, just after
the MYO10 FERM domain. In cells,GFP–MYO10–BioID localized
to and biotinylated proteins at filopodia tips (Fig. 1C). We purified
biotinylated proteins in cells expressing GFP–MYO10 (negative
control) or GFP–MYO10–BioID using streptavidin pull-downs
(Fig. 1D) and performed mass spectrometry analyses. Somewhat
unexpectedly, this approach identified very few proteins, perhaps due
to the slow kinetics of the biotin ligase used (Fig. 1E; Table S2).
Nevertheless, when comparing our GFP pulldown and BioID
datasets, only two proteins, MYO10 itself and lamellipodin
(RAPH1), were identified consistently as enriched with MYO10
overcontrols (Fig. 1E). Western blot analyses confirmed that RAPH1
co-purifies with GFP–MYO10
FERM
and that RAPH1 is biotinylated
in cells expressing GFP–MYO10–BioID (Fig. 1F,G). These results
led us to speculate that RAPH1 could be an MYO10 cargo.
MYO10 FERM is required for RAPH1 localization at filopodia
tips
RAPH1 is a member of the Mig-10/RIAM/lamellipodin (MRL)
protein family, with MIG-10 being the Caenorhabditis elegans
ortholog of RAPH1 (Coló et al., 2012). RAPH1 was previously
reported to localize to filopodia tips (Krause et al., 2004; Jacquemet
et al., 2019), but its contribution to filopodia function remains
unknown. Using structured illumination microscopy, we found that
RAPH1 specifically accumulates at filopodia tips where it
colocalizes with MYO10, whereas RIAM (also known as
APBB1IP) is uniformly distributed along filopodia (Fig. S1A). In
addition, live-cell imaging at high spatiotemporal resolution
indicated that RAPH1 closely follows MYO10 puncta at filopodia
tips throughout the filopodia life cycle (Fig. S1B; Movie 1).
Our mass spectrometry data indicated that the MYO10 FERM
domain binds to RAPH1. Therefore, we investigated the
requirement for MYO10-FERM to localize RAPH1 to filopodia
tips. We overexpressed an RFP-tagged MYO10 construct lacking
the FERM domain (MYO10
ΔF
) in cells (Miihkinen et al., 2021),
together with either RAPH1–GFP or VASP–GFP. It was not
necessary to suppress the expression of endogenous MYO10 here as
MYO10
ΔF
has a dominant-negative effect in U2-OS cells
(Miihkinen et al., 2021). Deleting the MYO10 FERM domain led
to a loss of RAPH1 accumulation at filopodia tips (Fig. 2A–D),
whereas VASP recruitment remained unaffected (Fig. 2A–D).
Importantly, we observe that the accumulation of endogenous
RAPH1 was also lost at the tip of MYO10
ΔF
filopodia (Fig. 2E,F).
In line with our results, others reported that the MYO10 FERM
domain was required for RAPH1 but not for VASP accumulation in
microspikes (Pokrant et al., 2023). Taken together, these findings
demonstrate that MYO10 and its FERM domain are required for
RAPH1 accumulation at filopodia tips. These results also suggest
that, despite containing multiple VASP-binding sites (Krause et al.,
2004), RAPH1 is not a prerequisite for VASP localization to
MYO10 filopodia.
RAPH1 directly interacts with MYO10
Next, we sought to identify the MYO10-binding domain(s) within
RAPH1. RAPH1 comprises several conserved domains, including a
Ras-association (RA) and a pleckstrin homology (PH) domain.
RAPH1 also contains known profilin-, VASP- and multiple putative
SH3-binding sites (Fig. 3A). Furthermore, previous work has
indicated that RAPH1 binds to talin-FERM via two N-terminal
talin-binding sites (Lee et al., 2009; Chang et al., 2014) (Fig. 3A;
Fig. S2A). As talin-FERM and MYO10-FERM share structural
similarities (Miihkinen et al., 2021), we speculated that RAPH1
could bind to MYO10-FERM via these talin-binding sites. To test
this hypothesis, we generated a RAPH1 deletion construct lacking
both talin-binding sites (Fig. S2B). Deleting both RAPH1 talin-
binding sites did not affect RAPH1 localization to filopodia tips
indicating that the RAPH1 talin-binding sites are not required for
MYO10 interaction (Fig. S2B).
Next, we generated four truncated RAPH1 constructs (named F1
to F4; Fig. 3A) and mapped their filopodia localization (Fig. 3B).
Somewhat surprisingly, the RAPH1 fragment F1 containing the PH
and the RA domains did not accumulate at filopodia tips. Indeed,
among the four constructs tested, only the RAPH1 F2 fragment,
which contains the profilin-binding sites, displayed an evident
accumulation at filopodia tips (Fig. 3B,C). This required an intact
MYO10 FERM domain and was lost in MYO10
ΔF
filopodia
(Fig. 3D,E), indicating that RAPH1 is recruited to filopodia tips via
this F2 region.
To validate MYO10–RAPH1 binding, we performed GFP-trap
experiments in MDA-MB-231 cells expressing GFP or GFP–
RAPH1
F2
. We chose MDA-MB-231 cells for their high
endogenous MYO10 protein levels (Jacquemet et al., 2019;
2016). MYO10 co-precipitated with GFP–RAPH1
F2
(Fig. 3F),
validating our microscopy-based assays. In addition, MYO10 was
pulled down from cell lysates using recombinant GST–RAPH1
F2
(Fig. S2C; Fig. 3G), and we detected binding between purified,
recombinant GST–RAPH1
F2
and His-tagged MYO10-FERM
proteins (Fig. 3H).
The RAPH1 region encompassed by the RAPH1 F2 fragment is
poorly characterized structurally. However, bioinformatic analyses
revealed that the amino acid sequence at residues 536–587 is very
well conserved (Fig. S3A–E). Expression of a GFP–RAPH1
536-587
construct (named GFP–RAPH1
F5
) showed that this construct could
accumulate at the tip of MYO10-containing filopodia (Fig. S3F).
Importantly, the deletion of the amino acid sequence 536 to 587 in
full-length RAPH1 was sufficient to nearly abolish RAPH1 binding
to MYO10 (Fig. 3I) and block RAPH1 localization and
accumulation at filopodia tips (Fig. 3J,K). The subcellular
localization of the RAPH1
Δ536-587
construct appeared otherwise
similar to that of full-length RAPH1 and accumulated to the cell
leading edge in lamellipodia (Fig. 3J).
Altogether, our data demonstrate a direct interaction between
RAPH1 and MYO10, and that RAPH1–MYO10 binding is required
for RAPH1 localization to filopodia tips. This interaction is
mediated by the MYO10 FERM domain and a previously
unexplored, conserved region within RAPH1 located after its PH
domain (Fig. 3A).
RAPH1 modulates filopodia formation and functions
Next, we investigated the contribution of RAPH1 to filopodia.
RAPH1 silencing with two independent siRNA oligonucleotides in
U2-OS cells expressing MYO10–GFP significantly reduced
MYO10-positive filopodia numbers (Fig. 4A,B). Importantly, this
phenotype could be rescued by expressing full-length RAPH1 but
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SHORT REPORT Journal of Cell Science (2023) 136, jcs260574. doi:10.1242/jcs.260574
Journal of Cell Science
not by expressing the RAPH1 construct lacking the F5 fragment
(Fig. 4C). In line with these results, others have found that RAPH1
knock-out reduces microspike formation in B16-F1 cells (Pokrant
et al., 2023). In addition, we observed that RAPH1 silencing also
slightly decreased filopodia length and, interestingly, in a small
proportion of RAPH1-silenced cells (below 1%), the filopodia tip
Fig. 1. See next page for legend.
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SHORT REPORT Journal of Cell Science (2023) 136, jcs260574. doi:10.1242/jcs.260574
Journal of Cell Science
complex collapsed, as observed by the dispersed localization of
MYO10 along the filopodia shaft (Fig. 4D). Although this
phenotype was rare, it was not observed in control cells.
RIAM and RAPH1 have been implicated in modulating integrin
activity (Lee et al., 2009). While this role is now well-established for
RIAM, it is more controversial for RAPH1 (Coló et al., 2012; Chang
et al., 2014; Lafuente et al., 2004; Watanabe et al., 2008). As the
MYO10 FERM domain is required to activate integrin at filopodia
tips (Miihkinen et al., 2021), we next investigated the role of
RAPH1 in modulating integrin activity at filopodia tips (Fig. S4).
Using SIM and filopodia mapping analyses, we found that integrin
activation at filopodia tip is comparable or slightly elevated in
RAPH1-depleted cells compared to that in control cells, indicating
that RAPH1 is not required for integrin activation at filopodia tips
(Fig. S4).
Finally, we explored the role of RAPH1 in modulating filopodia
dynamics in control and RAPH1-silenced U2-OS cells expressing
MYO10–GFP. Although the overall filopodia lifetime was
unaffected after RAPH1 depletion, MYO10 puncta moved
slightly faster and over longer distances than in control cells
(Fig. 4E,F), indicating that the filopodia tip complex is more
dynamic in RAPH1-depleted cells. The biological significance of
these differences remains, however, to be investigated.
Discussion and conclusions
Here, using two complementary mass spectrometry strategies, we
identify RAPH1 as a novel MYO10 interactor. Our results
demonstrate that (1) MYO10 is required to target RAPH1 to
filopodia tips, and (2) the MYO10–RAPH1 interaction contributes
to formation of filopodia containing MYO10. We propose that
MYO10 transports RAPH1 to filopodia tips, contributing to
filopodia stability via yet unknown mechanisms, possibly
involving RAPH1 interactions with other proteins such as VASP.
However, our data do not fully exclude the possibility that RAPH1
simply diffuses to filopodia and that MYO10 only contributes to
RAPH1 accumulation at filopodia tips without direct transport.
Testing this would require performing two-color single-molecule
imaging of MYO10 and RAPH1 to see whether these proteins move
toward filopodia tips together. However, we find that RAPH1 is not
very abundant in filopodia when the MYO10 FERM domain is
missing, suggesting that RAPH1 is likely to be actively transported
by MYO10.
RAPH1 is presumably in a complex with MYO10, VASP and
actin at filopodia tips. In this scenario, MYO10 could tether RAPH1
to filopodia tips using its motor domain, providing resistance against
the retrograde actin flow in filopodia (Bornschlögl, 2013; Lidke et al.,
2005). Once tethered, RAPH1 would cluster and increase VASP
activity by tethering VASP to the actin filaments (Hansen and
Mullins, 2015). Although we found that VASP molecules still
localize to filopodia tips in the absence of RAPH1, VASP activity will
likely be reduced, which could explain the shorter filopodia observed
in RAPH1-silenced cells. Therefore, we propose that MYO10-
mediated transport of RAPH1 to the filopodium tip is a feed-forward
mechanism that positively regulates MYO10 filopodia.
Interestingly, both MYO10 and RAPH1 have been implicated
separately as positive regulators of cancer cell migration and
invasion in similar contexts (Arjonen et al., 2014; Carmona et al.,
2016). In addition, MYO10 and RAPH1 knockout mice share
similar phenotypes, such as white belly patches due to defective
melanoblast migration (Heimsath et al., 2017; Law et al., 2013).
Therefore, it is tempting to speculate that the MYO10–RAPH1
interaction occurring at filopodia tips has strong relevance in health
and disease. Future work will investigate the contribution of the
MYO10–RAPH1 interaction in regulating cell migration in vivo.
MATERIALS AND METHODS
Cells
U2-OS osteosarcoma cells and MDA-MB-231 cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM; Sigma, D1152)
supplemented with 10% fetal bovine serum (FCS) (Biowest, S1860). U2-
OS cells were purchased from DSMZ (Leibniz Institute DSMZ-German
Collection of Microorganisms and Cell Cultures, Braunschweig, Germany;
ACC 785). MDA-MB-231 cells were provided by ATCC. The U2-OS
MYO10-GFP lines were generated by transfecting U2-OS cells
using Lipofectamine 3000 (Thermo Fisher Scientific), selected using
geneticin (Thermo Fisher Scientific; 400 μgml
−1
final concentration), and
sorted for green fluorescence using a fluorescence-assisted cell sorter
(FACS). All cell lines tested negative for mycoplasma. Cells were not
authenticated.
Antibodies and reagents
Mouse monoclonal antibodies used in this study were anti-β-actin [AC-15,
Merck, A1978; dilution for western blotting (WB), 1:1000], anti-His tag
(Thermo Fisher Scientific, MA1-21315; dilution for WB, 1:1000), and anti-
tubulin (DHSB, clone 12G10; dilution for WB, 1:1000). Rabbit polyclonal
antibodies used in this study were anti-RAPH1 [Thermo Fisher Scientific,
PA5-110270; dilution for WB, 1:1000; dilution for immunofluorescence
(IF) 1:100], anti-MYO10 (Novus Biologicals, 22430002; dilution for WB,
1:1000), and anti-GFP (Abcam, ab290). Biotinylated proteins were
detected using Streptavidin conjugated with Alexa Fluor™555 (for
immunofluorescence, dilution 1:100) or Alexa Fluor™647 (for western
blots, dilution 1:1000), both provided by Thermo Fisher Scientific (S21381
and S21374). The bovine plasma fibronectin was provided by Merck
(341631). DAPI (4’,6-diamidino-2-phenylindole, dihydrochloride) was
provided by Thermo Fisher Scientific (D1306).
Plasmids and transfection
U2-OS and MDA-MB-231 cells were transfected using Lipofectamine 3000
and the P3000TM Enhancer Reagent (Thermo Fisher Scientific) according
to the manufacturer’s instructions.
Fig. 1. Mass spectrometry analyses identify RAPH1 as a putative
MYO10 binder. (A,B) Mass spectrometry (MS) analysis of GFP–
MYO10
FERM
- and GFP–Talin
FERM
-binding proteins. Comparison of the
GFP–MYO10
FERM
dataset to GFP (A) and GFP–Talin
FERM
(B) datasets are
displayed as volcano plots where the fold-change enrichment is plotted
against the significance of the association (see Table S1 for the MS data).
The volcano plots were generated using VolcaNoseR (Goedhart and
Luijsterburg, 2020). (C) U2-OS cells transiently expressing GFP–MYO10–
BioID were plated on fibronectin (FN) in the presence of biotin for 24 h, fixed,
stained for biotinylated proteins (using streptavidin), F-actin, and DAPI, and
imaged using a spinning disk confocal (SDC) microscope. Scale bars:
25 µm (main); 5 µm (magnification). Note that only one cell in this field of
view expresses the GFP–MYO10–BioID construct. Images are
representative of three biological repeats. (D) U2-OS cells stably expressing
GFP–MYO10–BioID or GFP–MYO10 were plated on FN for 24 h in the
presence of biotin. Cells were then lysed, and biotinylated proteins were
purified using streptavidin beads. Recruited proteins were analyzed using
western blotting and MS (see Table S1 for the MS data). Western blots are
displayed (representative of five biological repeats). (E) Venn diagram
highlighting the overlap of MYO10-enriched proteins identified from the
indicated MS datasets. (F) GFP pulldown in U2-OS cells expressing GFP–
MYO10
FERM
, GFP–Talin
FERM
or GFP alone. RAPH1 recruitment to the bait
proteins was then assessed by western blotting (representative of three
biological repeats). (G) U2-OS cells stably expressing GFP–MYO10–BioID
or GFP-MYO10 were plated on FN for 24 h in the presence or absence of
biotin. Cells were then lysed, and biotinylated protein purified using
streptavidin beads. RAPH1 biotinylation was then assessed by western
blotting (representative of three biological repeats).
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Journal of Cell Science
Fig. 2. See next page for legend.
5
SHORT REPORT Journal of Cell Science (2023) 136, jcs260574. doi:10.1242/jcs.260574
Journal of Cell Science
The EGFPC1-hMyoX (MYO10-GFP) plasmid was Addgene plasmid
#47608 (deposited by Emanuel Strehler; Bennett et al., 2007). The
mScarlet-MYO10 (MYO10-RFP) construct was described previously
(Jacquemet et al., 2019) and is available on Addgene ( plasmid #145179).
The mScarlet-I-MYO10
ΔF
(MYO10
ΔF
-RFP) construct was previously
described (Miihkinen et al., 2021) and is also available on Addgene
(plasmid #145139). The GFP-VASP (mEmerald-VASP-N-10) plasmid was
Addgene plasmid #54297 (deposited by Michael Davidson). The GFP-
RIAM(1-666) construct was Addgene plasmid #80028 (deposited by
Chinten James Lim; Lee et al., 2013). The pcDNA3.1 MCS-BirA(R118G)-
HA construct was Addgene plasmid #36047 (deposited by Kyle Roux;
Roux et al., 2012). The RAPH1-GFP (EGFP-Lpd) plasmid was kindly
provided by Matthias Krause (King’s College London, UK).
The GFP–MYO10–BioID construct was generated as follows. Flanking
XbaI sites were introduced into BioID by PCR [5′-ATTAGATCTAGAG-
GATCCAAGGACAACACCGTGCCCCTG-3′,5′-ATTAGATCTAGAC-
TATGCGTAATCCGGTACATCGTAA-3′, template plasmid: BioID
pcDNA3.1 MCS-BirA(R118G)-HA] and the resulting amplicon was then
inserted into a unique XbaI site in the EGFPC1-hMyoX plasmid resulting in
an EGFP-MYO10-(stop codon)-BioID fusion gene. The stop codon
between MYO10 and BioID was then replaced with a codon encoding
valine (GTA) using a quick-change mutagenesis kit from Agilent and
following the manufacturer’s instructions.
The GFP–RAPH1
ΔTBS
[RAPH1 with amino acids (aa) 2–92 deleted]
construct was created by inserting a custom gene block (IDT) in the EGFP-
Lpd plasmid using the XhoI/HindIII sites. The sequence of the gene block is
provided below:
5′-ATTAGACTCGAGCCGCGATGTGCTCTATAGAGCAGGAGCT-
CAGCAGCATTGGTTCAGGAAACAGTAAGCGTCAAATCACAGAA-
ACGAAAGCTACTCAGAAATTGCCTGTTAGCCGACATACATTGAA-
ACATGGCACCTTGAAAGGATTATCTTCTTCATCTAATAGGATAGC-
TAAACCTTCCCATGCCAGCTACTCCTTGGACGACGTCACTGCAC-
AGTTAGAACAGGCCTCTTTGAGTATGGATGAGGCTGCTCAGCA-
ATCTGTACTAGAAGATACTAAACCCTTAGTAACTAATCAGCACA-
GAAGAACCGCGTCAGCAGGCACAGTGAGTGATGCTGAAGTACA-
CTCTATTAGTAATTCCTCCCATTCCAGCATCACTTCCGCAGCCTC-
CAGCATGGACTCTTTGGATATTGATAAAGTAACACGCCCTCAAG-
AGCTGGATTTGACACATCAAGGGCAGCCAATTACTGAGGAAGA-
ACAGGCAGCAAAATTGAAAGCTGAGAAGATCAGAGTTGCCCTA-
GAGAAAATTAAAGAGGCACAAGTGAAAAAGCTGGTGATCAGA-
GTCCACATGTCTGATGACAGTTCTAAAACAATGATGGTGGATGA-
GAGGCAGACAGTAAGACAAGTACTGGATAACCTGATGGACAAA-
TCCCACTGCGGTTATAGTTTAGACTGGTCACTGGTAGAAACCGT-
TTCTGAATTACAAATGGAGAGAATCTTTGAAGACCATGAAAACT-
TGGTTGAAAATCTTCTTAATTGGACAAGAGATAGCCAAAACAAG-
CTTATTAGA-3′.
The RAPH1 fragments F1 (RAPH1 aa 1–535), F2 (RAPH1 aa 535–868),
F3 (RAPH1 aa 868–1062), F4 (RAPH1 aa 1062–1250) and F5 (RAPH1 aa
536–587) constructs were purchased from GenScript. Briefly, the gene
fragments were synthesized using gene synthesis and cloned into
pcDNA3.1(+)-N-eGFP using the BamHI/XhoI sites. The GST–RAPH1
F2
construct (RAPH1 aa 535–868) was purchased from GenScript. The gene
fragment was synthesized using gene synthesis and cloned into pGEX-4T-1
using the BamHI/XhoI sites.
The GFP–RAPH1
Δ536-587
(RAPH1 aa 536-587 deleted) construct was
created by inserting a custom gene block (IDT) in the EGFP-Lpd plasmid
using the HindIII/KpnI sites. The sequence of the gene block is provided
below:
5′-AAGCTTATATTTATGGAGCGTATAGAAAAATATGCACTTTT-
CAAAAACCCACAGAATTATCTTTTGGGGAAAAAGGAAACAGCT-
GAGATGGCAGATAGAAACAAAGAAGTCCTCTTGGAGGAATGTT-
TTTGTGGAAGTTCTGTAACTGTACCAGAAATTGAAGGAGTCCTT-
TGGTTGAAGGATGATGGCAAGAAGTCCTGGAAAAAGCGTTATT-
TTCTCTTGCGAGCATCTGGTATCTACTATGTTCCCAAAGGAAAA-
GCAAAGGTCTCTCGGGATCTGGTGTGCTTTCTCCAGCTGGATCA-
TGTCAACGTTTATTATGGCCAGGACTATCGGAACAAATACAAAG-
CACCTACAGACTATTGTCTGGTGCTGAAGCATCCACAAATCCAG-
AAGAAATCTCAATATATCAAATACCTTTGTTGTGATGATGTGAG-
GACACTGCATCAGTGGGTCAATGGGATCCGCATTGCAAAGTATG-
GGAAGCAGCTCTATATGAACTACCAAGAAGCCTTGAAGAGGAC-
AGAGTCAGCCTATGATTGGACTTCCTTATCCAGCTCCAGCATTA-
AATCGGAAGAGTCCAGCAAGGCCAGAATGGAGTCTATGAATCG-
GCCCTACACTTCACTTGTGCCCCCTTTATCCCCGCAACCTAAGAT-
AGTCACCCCCTACACTGCTTCACAGCCTTCACCACCTCTACCTCC-
TCCGCCACCCCCACCTCCTCCTCCACCACCCCCTCCACCACCCCC-
TCCTCCCCCACTCCCCAGCCAGTCTGCACCTTCTGCAGGCTCAG-
CAGCCCCAATGTTCGTCAAGTACAGCACAATAACACGGCTACAG-
AATGCGTCTCAGCATTCAGGGGCCCTGTTTAAGCCGCCAACACC-
CCCAGTGATGCAGTCACAGTCAGTGAAGCCTCAGATCCTGGTA-
CC-3′.
siRNA-mediated gene silencing
The expression of RAPH1 was suppressed using 83 nM siRNA and
Lipofectamine 3000 (Thermo Fisher Scientific) according to the
manufacturer’s instructions. siRNAs used were AllStars Negative siRNA
control (cat. no. 1027418), RAPH1 siRNA #2 (Hs_RAPH1_2, SI00698642)
and RAPH1 siRNA #5 (Hs_RAPH1_5, SI04300982) provided by Qiagen.
SDS-PAGE and quantitative western blotting
Protein extracts were separated under denaturing conditions by SDS-PAGE
and transferred to nitrocellulose membranes using a Mini Blot Module
(Invitrogen, B1000). Membranes were blocked for 30 min at room
temperature using 1× StartingBlock buffer (Thermo Fisher Scientific,
37578). After blocking, membranes were incubated overnight with the
appropriate primary antibody (1:1000 in blocking buffer), washed three
times in PBS, and probed for 1 h using a fluorophore-conjugated secondary
antibody diluted 1:5000 in the blocking buffer. Membranes were washed
three times using PBS over 30 min and scanned using an iBright FL1500
imaging system (Invitrogen).
GFP-trap pulldown
Cells transiently expressing bait GFP-tagged proteins were lysed in a buffer
containing 20 mM HEPES pH 7.4, 75 mM NaCl, 2 mM EDTA, 1% NP-40,
as well as a cOmplete™protease inhibitor tablet (Roche, cat. no.
5056489001), and a phosphatase inhibitor mix (Roche cat. no.
04906837001). Lysates were then centrifuged at 15,000 gfor 5 min at
4°C. Clarified lysates were incubated with GFP-Trap magnetic or agarose
beads for 1 h at 4°C. Complexes bound to the beads were isolated by
centrifugation, washed three times with ice-cold lysis buffer, and eluted in
Laemmli reducing sample buffer for 10 min at 95°C.
Protein expression and purification
The BL-21(DE3) Escherichia coli strain (Merck, cat. no. 70954) was
transformed with plasmids encoding the relevant His-tagged or GST-tagged
Fig. 2. RAPH1 is recruited to filopodia tips in an MYO10-FERM-
dependent manner. (A–D) U2-OS cells expressing MYO10–RFP or
MYO10
ΔF
–RFP together with RAPH1–GFP (A) or VASP–GFP (B) were
plated on FN for 2 h, fixed, stained for F-actin and imaged using SIM. (A,B)
Representative maximum intensity projection (MIPs) are displayed. Yellow
arrows highlight filopodia tips. Scale bars: 20 µm (main); 2 µm
(magnifications). (C) Heatmap highlighting the sub-filopodial localization of
the proteins imaged in A and B generated from intensity profiles (n>300
filopodia per condition; three biological repeats). (D) The average RAPH1
and VASP staining intensity at filopodia tips measured in B are displayed as
box plots. (E,F) U2-OS cells expressing MYO10
WT
–RFP or MYO10
ΔF
–RFP
were plated on FN for 2 h, fixed, stained for F-actin and endogenous
RAPH1, and imaged using SIM. (E) A representative ROI is displayed.
Yellow arrows highlight filopodia tips. Scale bars: 2 µm. (F) The average
intensity of endogenous RAPH1 at filopodia tips is displayed as box plots
(n>175 filopodia per condition; three biological repeats). For all panels, the
data are shown as dot plots and Tukey boxplots. The whiskers (shown here
as vertical lines) extend to data points no further from the box than 1.5× the
interquartile range. The P-values were determined using a randomization
test. NS indicates no statistical difference between the mean values of the
highlighted condition and the control.
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Journal of Cell Science
Fig. 3. See next page for legend.
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SHORT REPORT Journal of Cell Science (2023) 136, jcs260574. doi:10.1242/jcs.260574
Journal of Cell Science
proteins. Bacteria were grown at 37°C in LB medium supplemented with
ampicillin (100 µg/ml; Fisher Bioreagents, cat. no. 10419313). Protein
expression was induced with IPTG (1 mM; Thermo Fisher Scientific, cat.
no. R0392) at 20°C. After 5 h, bacteria were harvested by centrifugation
(20 min at 6000 g) and resuspended in resuspension buffer [1× TBS, Pierce
Protease Inhibitor Tablet (Thermo Scientific, cat. no. A32963), 1× PMSF
(Sigma-Aldrich, cat. no. P7626), 0.05 mg/ml RNase (Roche, cat. no.
10109134001), 0.05 mg/ml DNase (Roche, cat. no. 11284932001)].
Bacteria were then lysed by adding BugBuster (Merck Millipore, cat. no.
70584-4) and a small spoonful of lysozyme (Thermo Scientific, cat. no.
89833). The suspension was mixed at 4°C for 30 min. Cell debris were then
pelleted by ultracentrifugation (at 20000 rpm, JA25.50 rotor) at 4°C for 1 h.
His-tagged MYO10 FERM was purified using a Protino Ni-TED 2000
packed column (Macherey Nagel, cat. no. 745120.25) according to the
manufacturer’s instructions. The protein was eluted in multiple 1 ml
fractions, supplemented with 1 mM AEBSF (Sigma-Aldrich, cat. no.
A8456), and kept at 4°C until needed (for up to 1 week). For GST-tagged
proteins, 600 µl of equilibrated glutathione–Sepharose 4B beads (GE
Healthcare, cat. no. 17-0756-01) was added to the supernatant and agitated
for 1 h at 4°C. Beads were collected and washed four times with TBS
supplemented with PMSF (1 mM). Protein-bound beads were stored at
−80°C until needed.
GST pull-down
GST and GST–RAPH1
F2
Sepharose beads were incubated with 10 mM His-
tagged MYO10
FERM
, and the mixture was rotated overnight at 4°C. Beads
were then washed four times with TBS supplemented with 1 mM PMSF.
Proteins bound to beads were then eluted in 2× Laemmli sample buffer at
80°C. Results were then analyzed by western blotting.
Proximity biotinylation
U2-OS cells stably expressing GFP–MYO10 or GFP–MYO10–BioID were
plated on fibronectin-coated plates ina medium containing 50 μM biotin for
24 h. After washing cells with cold PBS, cells were lysed, and debris were
removed by centrifugation (13,000 g, +4°C, 2 min). Biotinylated proteins
were then incubated with streptavidin beads (MyOne Streptavidin C1,
Invitrogen) for 1 h with rotation at +4°C. Beads were washed twice
with 500 μl wash buffer 1 [10% (w/v) SDS], once with 500 μl wash
buffer 2 [0.1% (w/v) deoxycholic acid, 1% (w/v) Triton X-100, 1 mM
EDTA, 500 mM NaCl, and 50 mM HEPES], and once with 500 μl
wash buffer 3 [0.5% (w/v) deoxycholic acid, 0.5% (w/v) NP-40, 1 mM
EDTA, and 10 mM Tris/HCl pH 7.4]. Proteins were eluted in 40 μlof
2× reducing sample buffer [100 mM Tris-HCl pH 6.5, 4% (w/v) SDS,
17.5% (v/v) glycerol, 3 mM bromophenol blue, 0.2 M dithiothreitol] for
10 min at 90°C.
Mass spectrometry analysis
Affinity-captured proteins were separated by SDS-PAGE and allowed to
migrate 10 mm into a 4–12% polyacrylamide gel. Following staining with
InstantBlue (Expedeon), gel lanes were sliced into five 2-mm bands. The
slices were washed using a solution of 50% 100 mM ammonium
bicarbonate and 50% acetonitrile until all blue color vanished. Gel slices
were washed with 100% acetonitrile for 5–10 min and then rehydrated in a
reducing buffer containing 20 mM dithiothreitol in 100 mM ammonium
bicarbonate for 30 min at 56°C. Proteins in gel pieces were then alkylated by
washing the slices with 100% acetonitrile for 5–10 min and rehydrated
using an alkylating buffer of 55 mM iodoacetamide in 100 mM ammonium
bicarbonate solution (protected from light, 20 min). Finally, gel pieces were
washed with 100% acetonitrile, followed by washes with 100 μl 100 mM
ammonium bicarbonate, after which slices were dehydrated using 100%
acetonitrile and fully dried using a vacuum centrifuge. Trypsin (0.01 μg/μl;
Promega, cat. no. V5111) was used to digest the proteins (37°C overnight).
After trypsinization, an equal amount of 100% acetonitrile was added, and
gel pieces were further incubated at 37°C for 15 min, followed by peptide
extraction using a buffer of 50% acetonitrile and 5% formic acid. The buffer
with peptides was collected, and the sample was dried using a vacuum
centrifuge. Dried peptides were stored at −20°C. Before liquid
chromatography electrospray ionization tandem mass spectrometry (LC-
ESI-MS/MS) analysis, dried peptides were dissolved in 0.1% formic acid.
The LC-ESI-MS/MS analyses were performed on a nanoflow HPLC system
(Easy-nLC1200, Thermo Fisher Scientific) coupled to the Orbitrap Fusion
Lumos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany)
equipped with a nano-ESI source. Peptides were first loaded on a trapping
column and subsequently separated inline on a 15 cm C18 column
(75 μm×15 cm, ReproSil-Pur 3 μm 120 Å C18-AQ, Dr Maisch HPLC
GmbH, Ammerbuch-Entringen, Germany). The mobile phase consisted of
water with 0.1% formic acid (solvent A) and acetonitrile/water [80:20 (v/v)]
with 0.1% formic acid (solvent B). Peptides were eluted with 40 min
method: from 8% to 43% of solvent B in 30 min, from 43% to 100% solvent
B in 2 min, followed bya wash for 8 min at 100% ofsolvent B. MS data was
acquired automatically by using Thermo Xcalibur 4.4 software (Thermo
Fisher Scientific). A data-dependent acquisition method consisted of an
Orbitrap MS survey scan of mass range 350–1750 m/zfollowed by HCD
fragmentation for the most intense peptide ions in a full speed mode with a
2.5 s cycle time.
Raw data from the mass spectrometer were submitted to the Mascot
search engine using Proteome Discoverer 1.5 (Thermo Fisher Scientific).
The search was performed against the human database SwissProt_2021_02,
assuming the digestion enzyme trypsin, a maximum of two missed
cleavages, an initial mass tolerance of 10 ppm (parts per million) for
precursor ions, and a fragment ion masstolerance of 0.020 Dalton. Cysteine
Fig. 3. RAPH1 is recruited to filopodia tips via a region located after its
PH domain. (A) Cartoon representation of RAPH1 domains. The
boundaries of the five fragments (F1 to F5) used in this study are
highlighted. (B) U2-OS cells expressing MYO10
WT
–RFP and one of the four
RAPH1 fragments (F1 to F4) were plated on FN for 2 h, fixed, stained for F-
actin and imaged using SIM. Representative maximum intensity projection
(MIPs) are displayed. Yellow arrows highlight filopodia tips. Scale bars:
10 µm (main); 2 µm (magnifications). (C) The preferential recruitment of the
four RAPH1 fragments to filopodia tips (F1 to F4) was assessed by
calculating an enrichment ratio (averaged intensity at filopodia tip versus
shaft; >415 filopodia per condition, three biological repeats). (D) U2-OS cells
expressing MYO10
WT
–RFP and the GFP–RAPH1
F2
(GFP-F2) were plated
on FN for 2 h, fixed, stained for F-actin, and imaged using SIM.
Representative MIPs are displayed. Yellow arrows highlight filopodia tips.
Scale bars: 10 µm (main); 2 µm (magnifications). (E) The preferential
recruitment of GFP–RAPH1
F2
to filopodia tips was assessed as in C (>427
filopodia per condition, three biological repeats). (F) GFP pulldowns in MDA-
MB-231 cells expressing GFP–RAPH1
F2
or GFP alone. Endogenous
MYO10 recruitment to the bait proteins was then assessed by western
blotting (representative of three biological repeats). (G) Pulldowns using
recombinant GST–RAPH1
F2
or GST alone in MDA-MB-231 cell lysates.
MYO10 binding to GST–RAPH1
F2
was then assessed by western blotting
(representative of three biological repeats). (H) Pulldowns using recombinant
GST–RAPH1
F2
or GST alone and recombinant His-tagged MYO10
FERM
.
MYO10
FERM
binding to GST–RAPH1
F2
was then assessed by western
blotting (representative of three biological repeats). (I) GFP pulldowns in
MDA-MB-231 cells expressing full-length RAPH1–GFP, RAPH1–GFP
lacking the F5 fragment (RAPH1
Δ536-587
) or GFP alone. Endogenous
MYO10 recruitment to the bait proteins was then assessed by western
blotting (representative of three biological repeats). The quantifications of
MYO10 recruitment to the bait protein are displayed as a SuperPlot where
individual repeats are color-coded (P-value calculated using a Welch’st-
test). Inputs in F, G and I, 5%. (J,K) U2-OS cells expressing MYO10–RFP
together with full-length RAPH1–GFP (J) or RAPH1–GFP lacking the F5
fragment (RAPH1
Δ536-587
) were plated on FN for 2 h, fixed, stained for F-
actin, and imaged using SIM. (J) Representative MIPs are displayed. Yellow
arrows highlight filopodia tips. Scale bars: 10 µm (main); 2 µm
(magnifications). (K) The average intensity and preferential recruitment of
the two RAPH1 constructs to filopodia tips are displayed as box plots (n>800
filopodia per condition; three biological repeats). For all panels, the data are
shown as dot plots and Tukey boxplots (except I). The whiskers (shown here
as vertical lines) extend to data points no further from the box than 1.5× the
interquartile range. The P-values were determined using a randomization
test (except panel I). NS indicates no statistical difference between the mean
values of the highlighted condition and the control.
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SHORT REPORT Journal of Cell Science (2023) 136, jcs260574. doi:10.1242/jcs.260574
Journal of Cell Science
carbamidomethylation was set as a fixed modification, and methionine
oxidation was set as a variable modification.
To generate the MYO10–BioID dataset, five biological replicates
were combined. Proteins enriched at least twofold in GFP–MYO10–
BioID over GFP-MYO10 (based on spectral count) and detected with
over five spectral counts (across all repeats) were considered putative
MYO10-binding proteins. To generate the MYO10
FERM
and TLN1
FERM
datasets, two biological replicates were combined. Proteins enriched at least
twofold in MYO10
FERM
over GFP and over TLN1
FERM
(based on spectral
count) and detected with more than ten spectral counts (across both repeats)
Fig. 4. See next page for legend.
9
SHORT REPORT Journal of Cell Science (2023) 136, jcs260574. doi:10.1242/jcs.260574
Journal of Cell Science
were considered putative MYO10-binding proteins. The fold-change
enrichment and the significance of the association used to generate
the volcano Plots (Fig. 1A,B) were calculated directly in Proteome
Discoverer.
Light microscopy setup
The spinning-disk confocal microscope used was a Marianas spinning-disk
imaging system with a Yokogawa CSU-W1 scanning unit on an inverted
Zeiss Axio Observer Z1 microscope controlled by SlideBook 6 (Intelligent
Imaging Innovations, Inc.). Images were acquired using either an Orca Flash
4 sCMOS camera (chip size 2048×2048; Hamamatsu Photonics) or an
Evolve 512 EMCCD camera (chip size 512×512; Photometrics). The
objective used was a 100× oil (NA 1.4 oil, Plan-Apochromat, M27)
objective.
The structured illumination microscope (SIM) used was DeltaVision
OMX v4 (GE Healthcare Life Sciences) fitted with a 60× Plan-Apochromat
objective lens, 1.42 NA (immersion oil RI of 1.516) used in SIM
illumination mode (five phases×three rotations). Emitted light was collected
on a front-illuminated pco.edge sCMOS (pixel size 6.5 mm, readout speed
95 MHz; PCO AG) controlled by SoftWorx.
The confocal microscope used was a laser scanning confocal microscope
LSM880 (Zeiss) equipped with an Airyscan detector (Carl Zeiss) and a 40×
water (NA 1.2) or 63× oil (NA 1.4) objective. The microscope was
controlled using Zen Black (2.3), and the Airyscan was used in standard
super-resolution mode.
Quantification of filopodia numbers and dynamics
For the filopodia formation assays, cells were plated on fibronectin-coated
glass-bottom dishes (MatTek Corporation) for 2 h. Samples were fixed
for 10 min using a solution of 4% PFA, then permeabilized using a solution
of 0.25% (v/v) Triton X-100 for 3 min. Cells were then washed with PBS
and quenched using a solution of 1 M glycine for 30 min. Samples were
then washed three times in PBS and stored in PBS containing SiR-actin
(100 nM; Cytoskeleton; catalog number: CY-SC001) at 4°C until imaging.
Just before imaging, samples were washed three times in PBS. Images were
acquired using a spinning-disk confocal microscope (100× objective). The
number of filopodia per cell was manually scored using Fiji (Schindelin
et al., 2012).
To study filopodia stability, U2-OS cells expressing MYO10–GFP were
plated on fibronectin for at least 2 h before the start of live imaging (pictures
taken every 5 s at 37°C, on an Airyscan microscope, using a 40× objective).
All live-cell imaging experiments were performed in normal growth
medium, supplemented with 50 mM HEPES, at 37°C and in the presence of
5% CO
2
. Filopodia lifetimes were then measured by identifying and
tracking all MYO10 spots using the Fiji plugin TrackMate (Tinevez et al.,
2017; Ershov et al., 2022). In TrackMate, the custom Stardist detector and
the simple LAP tracker (Linking max distance=1 micron, Gap-closing max
distance=0 microns, Gap-closing max frame gap=0 micron) were used. The
StarDist 2D model used was trained for 200 epochs on 11 paired image
patches [image dimensions: (512, 512), patch size: (512,512)] with a batch
size of 2 and a mean absolute error (MAE) loss function, using the StarDist
2D ZeroCostDL4Mic notebook (von Chamier et al., 2021; Schmidt et al.,
2018). The training was accelerated using a Tesla K80 GPU.
Generation of filopodia maps and analysis of filopodia length
U2-OS cells transiently expressing the constructs of interest were plated on
high tolerance glass-bottom dishes (MatTek Corporation, coverslip #1.7)
pre-coated first with poly-L-lysine (10 µg/ml, 1 h at 37°C; Sigma-Aldrich,
cat. no. A-005-M) and then with bovine plasma fibronectin (10 µg/ml, 2 h at
37°C). After 2 h, samples were fixed and permeabilized simultaneously
using a solution of 4% (w/v) PFA and 0.25% (v/v) Triton X-100 for 10 min.
Cells were then washed with PBS, quenched using a solution of 1 M glycine
for 30 min, and, when appropriate, incubated with the primary antibody for
1 h (1:100). After three washes, cells were incubated with a secondary
antibody for 1 h (1:100). Samples were then washed three times and
incubated with SiR-actin (100 nM in PBS; Cytoskeleton, cat. no. CY-
SC001) at 4°C until imaging (minimum length of staining, overnight at 4°C;
maximum length, 1 week). Just before imaging, samples were washed three
times in PBS and mounted in Vectashield (Vector Laboratories).
To map the localization of each protein within filopodia, images were first
processed in Fiji (Schindelin et al., 2012), and data were analyzed using R
software as previously described (Jacquemet et al., 2019). Briefly, in Fiji,
the brightness and contrast of each imagewere automaticallyadjusted using,
as an upper maximum, the brightest cellular structure labeled in the field of
view. In Fiji, line intensity profiles (1-pixel width) were manually drawn
from filopodia tip to base (defined by the intersection of the filopodia and
the lamellipodium). The intensity profile lines were drawn from a merged
image to avoid any bias in the analysis. All visible filopodia in each image
were analyzed and exported for further analysis (export was performed
using the ‘Multi Plot’function). For each staining, line intensity profiles
were then compiled and analyzed in R software. To homogenize filopodia
length, each line intensity profile was binned into 40 bins (using the median
value of pixels in each bin and the R function ‘tapply’). The map of each
protein of interest was created by averaging hundreds of binned intensity
profiles. The length of each filopodium analyzed was directly extracted from
the line intensity profiles.
The preferential recruitment of protein to filopodia tips or shafts was
assessed by calculating an enrichment ratio where the averaged intensity of
the signal at the filopodia tip (bin 1–6) was divided by the averaged intensity
at the filopodia shaft (bin 7–40).
Quantification and statistical analysis
Randomization tests were performed using the online tool
PlotsOfDifferences (https://huygens.science.uva.nl/PlotsOfDifferences/)
(Goedhart, 2019 preprint). Dot plots were generated using PlotsOfData
(Postma and Goedhart, 2019). Volcano Plots were generated using
VolcaNoseR (Goedhart and Luijsterburg, 2020). Superplots were
generated using SuperPlotsOfData (Goedhart, 2021). All numerical raw
data and associated statistical analyses (including effect size) are available in
Table S2. The ImageJ macro and the R code used to generate the filopodia
maps were previously described and are available on GitHub (https://github.
com/guijacquemet/FiloMAP).
Acknowledgements
We thank J. Siivonen and P. Laasola for technical assistance and H. Hamidi for
editing the manuscript. The Cell Imaging and Cytometry Core facility (Turku
Fig. 4. RAPH1 supports filopodia formation and the stability of the
filopodia tip complex. (A) Efficiency of siRNA-mediated RAPH1 silencing
using two different siRNA oligonucleotides in U2-OS cells (representative of
three biological repeats). (B) RAPH1-silenced U2-OS cells transiently
expressing MYO10–GFP were plated on FN for 2 h, fixed, and the number
of MYO10-positive filopodia per cell was quantified (n>93 cells per condition,
three biological repeats). Scale bars: 25 µm (main); 5 µm (magnifications).
(C) RAPH1-silenced U2-OS cells transiently expressing MYO10–RFP
together with GFP, RAPH1
FL
–GFP or RAPH1
Δ536-587
–GFP were plated on
FN for 2 h, fixed, and the number of MYO10-positive filopodia per cell was
quantified (n>60 cells per condition, three biological repeats). Scale bars:
25 µm (main); 5 µm (magnifications). (D) RAPH1-silenced U2-OS cells
transiently expressing MYO10–GFP were plated on FN, stained for F-actin,
and imaged using SIM. Representative maximum intensity projection (MIPs)
are displayed. Scale bars: 20 µm (main); 2 µm (magnifications).
Quantifications of filopodia length from SIM images are displayed (n>530
filopodia per condition; three biological repeats). (E,F) RAPH1-silenced U2-
OS cells transiently expressing MYO10–GFP were plated on FN and imaged
live using an Airyscan confocal microscope (1 picture every 5 s over 20 min).
For each condition, MYO10-positive particles were automatically tracked. (E)
The average MYO10 track duration per cell is displayed (three biological
repeats, n>17 cells per condition). (F) The average speed and the total
distance traveled by MYO10 spots are displayed (n>9600 filopodia; three
biological repeats). For all panels, the data are shown as dot plots (except F)
and Tukey boxplots. The whiskers (shown here as vertical lines) extend to
data points no further from the box than 1.5× the interquartile range. The
P-values were determined using a randomization test. NS indicates no
statistical difference between the mean values of the highlighted condition
and the control.
10
SHORT REPORT Journal of Cell Science (2023) 136, jcs260574. doi:10.1242/jcs.260574
Journal of Cell Science
Bioscience, University of Turku, Åbo Akademi University, and Biocenter Finland)
and Turku Bioimaging are acknowledged for services, instrumentation, and
expertise. Mass spectrometry analyses were performed at the Turku Proteomics
Facility supported by Biocenter Finland.
Competing interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: J.I., G.J.; Methodology: A.P., M.M., G.J.; Software: G.J.;
Validation: G.J.; Formal analysis: A.P., M.M., R.S., M.L.B.G., B.T.G., G.J.;
Investigation: A.P., M.M., S.G., R.S., M.L.B.G., N.J.B., J.I., G.J.; Resources: N.J.B.,
B.T.G., J.I., G.J.; Data curation: G.J.; Writing - original draft: G.J.; Writing - review &
editing: A.P., M.M., S.G., R.S., M.L.B.G., B.T.G., J.I., G.J.; Visualization: A.P., M.M.,
J.I., G.J.; Supervision: J.I., G.J.; Project administration: J.I., G.J.; Funding
acquisition: J.I., G.J.
Funding
This study was supported by the Academy of Finland (G.J., 338537, and J.I, 325464;
346131), the Sigrid Juselius Foundation (J.I.), the Cancer Society of Finland (G.J.
and J.I.), and Åbo Akademi University Research Foundation (G.J., CoE CellMech)
and by Solution for Health strategic funding to Åbo Akademi University (G.J.) and the
Finnish Cancer Institute (K. Albin Johansson Professorship to J.I.). M.M. has been
supported by the Drug Research Doctoral Programme, the University of Turku
foundation, Maud Kuistila Foundation, Instrumentarium Foundation, Lounais-
Suomen Syo
pa
yhdistys, K. Albin Johansson’s Foundation, and Ida Montin
Foundation. B.T.G. was supported by the Biotechnology and Biological Sciences
Research Council grant BB/S007245/1. A.P. was supported by the K. Albin
Johanssons, the Åbo Akademi Doctoral, and the Swedish Cultural Foundations.
This research was supported by InFLAMES Flagship Programme of the Academy
of Finland (decision numbers: 337531 and 337530). Open access funding provided
by the University of Turku. Deposited in PMC for immediate release.
Data availability
All relevant data can be found within the article and its supplementary information.
The corresponding authors, upon reasonable request, will share the raw data used
to reach the conclusions reported in this paper.
First Person
This article has an associated First Person interview with the first authorof the paper.
Peer review history
The peer review history is available online at https://journals.biologists.com/jcs/
lookup/doi/10.1242/jcs.260574.reviewer-comments.pdf
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Fig. S1. RAPH1, but not RIAM, accumulates at the tip of MYO10 filopodia. (A) U2-OS
cells expressing MYO10-RFP and RAPH1-GFP or RIAM-GFP were plated on FN for 2 h,
fixed, stained for F-actin, and imaged using structured illumination microscopy (SIM).
Representative maximum intensity projections (MIPs) are displayed. Scale bars: (main) 20
µm; (inset) 2 µm. (B) U2-OS cells expressing MYO10-RFP and RAPH1-GFP were imaged
live at high spatiotemporal resolution using an Airyscan confocal microscope. A single
time point (upper panel, scale bar: 2 µm) and a kymograph (lower panel) are displayed.
J. Cell Sci.: doi:10.1242/jcs.260574: Supplementary information
Journal of Cell Science • Supplementary information
Fig. S2. RAPH1 putative talin-binding sites do not contribute to its filopodia tip
localization. (A) Alignment of the RAPH1 and RIAM talin-binding sites (TBS). (B) U2-OS
cells expressing MYO10WT-RFP and GFP-RAPH1 lacking both TBS (GFP-RAPH1∆TBS) were
plated on FN for 2 h, fixed, stained for F-actin, and imaged using SIM. Representative MIPs
are displayed. Yellow arrows highlight filopodia tips; scale bars: (main) 10 µm; (inset) 2 µm.
(C) Recombinant GST-RAPH1F2 and GST were produced in bacteria and subsequently
purified using Glutathione agarose beads. Produced proteins were run on a polyacrylamide
gel and stained using coomassie.
J. Cell Sci.: doi:10.1242/jcs.260574: Supplementary information
Journal of Cell Science • Supplementary information
J. Cell Sci.: doi:10.1242/jcs.260574: Supplementary information
Journal of Cell Science • Supplementary information
Fig. S3. The MYO10 binding site in RAPH1 is highly conserved. (A) Cartoon of
RAPH1 domain structure highlighting the four truncated RAPH1 constructs, F1 to F5.
(B) Structural model of RAPH1 generated using AlphaFold (Jumper et al., 2021;
Varadi et al., 2022) and colored as in (A). The majority of RAPH1 protein is predicted
to be unstructured. (C) The F2 region of RAPH1 was shown to harbor the MYO10
binding site. (D-E) The F2 region of RAPH1 is colored based on the evolutionary
conservation of RAPH1 as determined using the ConSurf server (Ashkenazy et al.,
2016). The main chain is shown as spheres (D) and cartoons (E). Highly conserved
regions are shown in purple. The region 536-587 is highly conserved. The poly-proline
motifs are also identified using this approach and are highlighted. (E) U2-OS cells
expressing MYO10WT-RFP and GFP-RAPH1 536-587 were plated on FN for 2 h,
fixed, stained for F-actin, and imaged using SIM. Representative MIPs are displayed.
Yellow arrows highlight filopodia tips; scale bars: (main) 10 µm; (inset) 2 µm.
J. Cell Sci.: doi:10.1242/jcs.260574: Supplementary information
Journal of Cell Science • Supplementary information
Fig. S4. RAPH1 is not required for β1-integrin activation at filopodia tips. (A-B)
RAPH1-silenced U2-OS cells expressing MYO10-GFP were plated on FN for 2 h, stained
for active β1-integrin (12G10), and imaged using SIM. Representative MIPs are displayed;
scale bars: (main) 20 µm; (inset) 2 µm. (B) The average intensity of 12G10 at filopodia tips
was measured from line intensity profiles and displayed as boxplots (n > 400 filopodia,
three biological repeats). P-values were determined using a randomization test. NS
indicates no statistical difference between the mean values of the highlighted condition
and the control.
Table S1. This file contains the MS analysis of GFP-MYO10FERM and GFP-
TalinFERM binding proteins and the MS analysis of biotinylated proteins in cells
expressing GFP-MYO10-BioID or GFP-MYO10.
Table S2. This file contains the raw numerical values of the datasets used in this
study and their statistical analyses.
Click here to download Table S1
Click here to download Table S2
J. Cell Sci.: doi:10.1242/jcs.260574: Supplementary information
Journal of Cell Science • Supplementary information
