Journal of Proteomics 240 (2021) 104222

Contents lists available at ScienceDirect

Journal of Proteomics
journal homepage: www.elsevier.com/locate/jprot

Proteomic analysis of Caenorhabditis elegans wound model reveals novel

molecular players involved in repair
Murugesan Pooranachithra a, Chelladurai Satheesh Kumar a, James Prabhanand Bhaskar b,
Krishnan Venkateswaran b, Velayutham Ravichandiran c, Krishnaswamy Balamurugan a, *
a
Department of Biotechnology, Science Campus, Alagappa University, Karaikudi 630 003, Tamil Nadu, India
b
ITC - Life Sciences and Technology Centre, Peenya Industrial Area, 1st Phase, Bangalore 560058, Karnataka, India
c
National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India

A R T I C L E I N F O A B S T R A C T

Keywords: Wound repair is a multistep process which involves coordination of multiple molecular players from different cell
Wound repair types and pathways. Though the cellular processes that are taking place in order to repair damage is already
2-D GE known, molecular players involved in crucial pathways are still scarce. In this regard, the present study intends to
LCMS/MS
uncover crucial players that are involved in the central repair events through proteomics approach which
PPI network analysis
included 2-D GE and LC-MS/MS using Caenorhabditis elegans wound model. Initial gel-based 2-D GE and
Mitochondrial fission
following protein-protein interaction (PPI) network analyses revealed active role of calcium signaling, acetyl­
choline transport and serotonergic neurotransmitter pathways. Further, gel-free LC-MS/MS and following PPI
network analyses revealed the incidence of actin nucleation at the initial hours immediately after injury. Further
by visualizing the PPI network and the interacting players, pink-1, a mitochondrial Serine/threonine-protein
kinase which is known to regulate mitochondrial dynamics, was found to be the central player in facilitating
the mitochondrial fission and its role was further verified using qPCR analysis and pink-1 transgenic worms.
Overall, the study delivers new insights from crucial regulatory pathways and central players involved in wound
repair using high throughput proteomic approaches and the mass spectrometry Data (PXD024629/PXD024744)
are available via ProteomeXchange.
Significance:

• Though, C. elegans is being explored as a wound model for more than a decade, global proteomic
changes are not looked at yet upon injury and following repair. In this regard, the present study was
carried out in C. elegans to uncover new insights from wound repair through global proteomic
analysis.

Abbreviations: 2-D GE, 2-dimensional gel electrophoresis; ACN, Acetonitrile; AmBiC, Ammonium bicarbonate; ANOVA, analysis of variance; CBB, Coomassie
Brilliant Blue; DTT, Dithiothreitol; GO, Gene Ontology; IAA, Iodoacetamide; IPG, Immobilized pH Gradient; KEGG, Kyoto Encyclopaedia of Genes and Genomes; LC-
MS/MS, Liquid Chromatography Tandem Mass Spectrometry; MALDI, Matrix-Assisted Laser Desorption/Ionization; MCODE, Molecular complex detection; NGM,
Nematode Growth Medium; NHS, National Health Service; NIH, National Institutes of Health; OP50, Escherichia coli OP50; PMSF, Phenyl Methyl Sulfonyl Fluoride;
PPI, Protein-Protein Interaction; SDS PAGE, Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis; STRING, Search Tool for the Retrieval of Interacting
Genes; TOF, Time Of Flight; UW, Unwounded; W, Wounded; W1, Worms wounded by vortex for 1 min; W2, Worms wounded by vortex for 3 min; W3, Worms
wounded by vortex for 5 min; W4, Worms wounded by vortex for 7 min; W5, Worms wounded by vortex for 10 min; acbp-1, Acyl-CoA-binding protein homolog 1; act-
1, Actin-1; atp-2, ATP synthase subunit beta, mitochondrial; C34D4.1, Protein maph-9; cpna-1, Copine family protein 1; cul-1, Cullin-1; dys-1, Dystrophin-1; fat-3, FAT
Atypical Cadherin 3; gad-1, Glutamate decarboxylase 1; gcy-9, Receptor-type guanylate cyclase 9; gls-1, Germline survival defective-1; mks-3, MecKel-Gruber Syn­
drome (MKS) homolog; mlc-1, Myosin regulatory light chain 1; osm-1, Intraflagellar transport protein; pink-1, mitochondrial serine/threonine-protein kinase; pkc-2,
Protein kinase C-like 2; sas-4, Spindle assembly abnormal protein 4; spdl-1, Spindly-like protein spdl-1; tag-173, Transket_pyr domain-containing protein; tbb-1,
Tubulin beta chain; tln-1, Talin; ubq-1, Polyubiquitin-A; unc122, Olfactomedin-like domain-containingprotein; unc-15, Paramyosin; unc-26, Homeobox protein unc-4;
unc-54, Myosin-4; unc-82, Protein kinase domain-containing protein; unc-87, Protein unc-87; Y40C5A.1, ZZ-type domain-containing protein; ZK632.12, Uncharac­
terized protein.
* Corresponding author.
E-mail addresses: pooranachithra@gmail.com (M. Pooranachithra), yesiamcsk@gmail.com (C. Satheesh Kumar), James.Bhaskar@itc.in (J.P. Bhaskar), krishnan.
venkateswaran@itc.in (K. Venkateswaran), directorniperkolkata@gmail.com (V. Ravichandiran), bsuryar@yahoo.com (K. Balamurugan).

https://doi.org/10.1016/j.jprot.2021.104222
Received 21 November 2020; Received in revised form 12 March 2021; Accepted 31 March 2021
Available online 5 April 2021
1874-3919/© 2021 Elsevier B.V. All rights reserved.
M. Pooranachithra et al.
• For this purpose, 2-D GE and LC-MS/MS analysis was carried out in unwounded and wounded
samples and the PPI network constructed from the identified proteins included 2683 non redundant
nodes which is actually 1/10th of the total nodes present in C. elegans whole PPI network available
in the database as shown in the figure below. This is an added value of the present study which
resulted with fruitful insights into crucial regulatory players and pathways that are regulated
during wound repair.
1. Introduction
Wound healing is a process that involves spatial and temporal syn­
chronisation of numerous cell types that possess distinct roles during
each of the phases of Haemostasis/ coagulation, inflammation, Prolif­
eration/ re-epithelialization, and remodelling [1]. This is one of the
most complex processes in the human body in which the underlying
cellular and molecular mechanisms of the repair and failure of the same
is poorly understood [2]. Owing to a significant socioeconomical burden
of the prevalence and recurrence of the wounds, it was stated that there
is an urgent requirement for the improved understanding of the bio­
logical and clinical mechanisms of repair. Moreover, application of
emerging research technologies is also essential for elucidating the un­
derlying cellular and molecular basis of acute and pathological repair
[3] and hence the present study was intended to identify the key regu­
latory players involved in wound repair by employing standard prote­
omic analyses tools such as 2-dimentional gel electrophoresis (2-D GE)
and Liquid Chromatography Mass Spectrometry (LCMS/MS).
As the process of repair after injury is fundamental to all multicel­
lular organisms, understanding them across the phyla is expected to
improve our understanding of the process in humans [4]. In this regard,
Caenorhabditis elegans from the phylum Nematoda was opted as wound
model in the present study whereas interestingly, the model was re­
ported as a tractable one to dissect the epidermal epithelial response to
injury [5]. Epidermal wounding of C. elegans involves two different
concurrent pathways which includes cutaneous innate immune response
and wound closure. The innate immune response involves up-regulation
of a suite of antimicrobial peptides such as nlp-29 and nlp-31 through a G
Protein–Coupled Receptor (GPCR)/ Mitogen-Activated Protein Kinase
(MAPK) cascade [6] and the wound closure process involves transient
receptor potential M class (TRPM) channel/ Ca2+ pathway mediated
actin polymerisation/ actin cytoskeleton rearrangement [7–9]. More­
over, crucial molecular events involved in wound healing such as ROS/
Ca2+ mediated wound response, F-actin dynamics mediated wound
closure and signaling cascades such as hippo signaling, Notch signaling,
Wnt/ β-catenin, TGF- β signaling, etc. were reported to be conserved in
C. elegans (See Suppl. Table S1 for more information).
Among the fascinating findings in wound research using C. elegans, a
recent study reported that the localised response after injury was un­
covered to be mediated by mitochondrial fragmentation [10] wherein it
was also proven to be applicable across the phyla [11–13] which in turn
endorse the applicability of the model in wound research. Moreover,
there are number of molecular players involved in mitochondrial dy­
namics that are conserved in C. elegans including PINK-1, a PTEN-
induced Kinase which was reportedly a key molecular player of
mitophagy [14] that promotes mitochondrial fission upon over expres­
sion [15].
2. Materials and methods
2.1. C. elegans strains
Wild type (N2) C. elegans strain and PINK-1 transgenic worm named
BR4006 [pink-1(tm1779) II; byEx655 (pink-1p::pink-1::GFP + myo-2p::
mCherry + herring sperm DNA) obtained from CGC, USA were used in
the present study. In BR4006, pink-1 translational GFP (C-terminal GFP)
fusion was generated by cloning the complete pink-1 genomic fragment
2
Journal of Proteomics 240 (2021) 104222
(3191 bp) with a 6-kb upstream promoter region and into pPD117.01.
Nematodes were maintained on nematode growth medium (NGM)
plates seeded with standard laboratory food source Escherichia coli OP50
(OP50) at 20 ◦ C (Wild type) and 15 ◦ C (Transgenic worm). Gravid ani­
mals were bleached by exposing them to 5 M KOH and commercial
bleach (1,1) for a minute to obtain only the eggs. KOH and Bleach
treated worms were later transferred to a new NGM plate and grown to
collect synchronous population. For experiments, age synchronised
young adult hermaphrodite animals were used in triplicates [16].
2.2. Glass wool mediated wounding of voluminous C. elegans
For wounding of greater number of worms at a stretch, glass wool
mediated wounding protocol was used [17]. Briefly, worms were vor­
texed for 1, 3, 5, 7 and 10 min for W1 to W5, respectively. The worms
were then washed gently with M9 buffer and scored for injured, unin­
jured and disintegrated worms under stereo microscope (Nikon
SMZ1000, Japan) to assess the success rate of injury [18].
2.3. Locomotion assay
Unwounded and wounded (W1-W5) worms (~50 numbers) were
transferred to NGM plates where movement of the worms was observed
under stereo microscope for 1 min along with live recording of loco­
motion of the worms [19].
2.4. Analysis of recovery after injury
The wounded worms (W1-W5) (~50 numbers) were transferred to
separate NGM plate and allowed for recovery by placing them in incu­
bator at 20 ◦ C for 24 h [20]. Following incubation after 24 h, the effect of
wound healing/ recovery process was monitored under stereo micro­
scope. Movement of worms from original to the nearby OP50 lawn was
considered as recovery. Failure of movement and decaying of the
worm’s cellular contents was considered as failure of recovery and dead,
respectively.
2.5. Survival assay
Assay was performed as described in a previous study with slight
modifications [21]. Briefly, each of the well in 24 well plate containing
10 worms (UW/W) were inoculated with 20% of OP50 food source and
survival was counted manually under stereo microscope at 4 h time
intervals for 72 h [18].
2.6. Protein isolation and estimation
Equal quantity of unwounded (control) and wounded worms (~8000
numbers) were stored in 8 M Urea buffer followed by the addition of 1
mM final concentration of Phenyl Methyl Sulfonyl Fluoride (PMSF, a
serine protease inhibitor). The samples were sonicated for about 2 min
(pulse 5 S On; 10 S Off) and centrifuged at 7500 rpm for 5 min at 4 ◦ C.
The supernatant was collected and stored at − 20 ◦ C until experiment.
Quantification of proteins was done by Bradford method using bovine
serum albumin as standard. At least 1 mg and 200 μg of protein were
used for 2-Dimensional Gel Electrophoresis (2-DGE) and Liquid chro­
matography tandem mass spectrometry (LC-MS/MS), respectively.
M. Pooranachithra et al.
2.7. 2-D GE
2-D GE was performed in triplicates according to Balasubramanian
et al. 2016 with minor modifications [16]. Briefly, 1 mg of protein was
purified using 2-D clean up kit (GE healthcare) to remove the non-
specific biological contaminants such as lipids, nucleic acids, and salts
and re-suspended in sample buffer containing urea and thiourea. Later,
the protein was loaded onto an immobilized pH gradient (IPG) strips (24
cm, pH 3–10 NL, GE Healthcare) and rehydrated for 12 h at room
temperature. Subsequently, the sample was subjected to first dimension
electrophoresis based on isoelectric point (PI) through isoelectric
focusing (IEF) using Ettan™IPGphor 3 isoelectric focusing system. Later,
the strips were allowed for second dimension electrophoresis followed
by equilibration with dithiothreitol (DTT) (10 mg/ml) and iodoaceta­
mide (IAA) (25 mg/ml). Among the various visible stains including
Coomassie Brilliant Blue G-250 (CBB) and silver nitrate (SN), the gels
were stained with colloidal Coomassie Brilliant Blue (CCB) after elec­
trophoresis by comparing the advantages and limitations of each of
them. Though SN staining was reported as highly sensitive and much
more suitable for MS analysis, CCB staining becomes more advantageous
owing to its linear dynamic range. CCB offers better quantitative dif­
ferences in protein expression than that of silver staining which makes it
easier to detect in 2-D GE gel images [22]. Hence, CCB staining was
opted in the present study where high protein loading (1 mg) assisted in
overcoming the issue of sensitivity compared to SN staining.
2.8. Spot analysis after 2-D GE
Stained gels were subjected to Gel scanner-III and the resulting image
was analysed with Image Master 2-D platinum version 7 software (GE
Healthcare). Briefly, for the purpose of recognising majority of spots
after automatic spot detection, manual editing of spots was done using
the tool kit available with the software including add, split, create,
remove and merge spots. Later, classes were created with triplicate gels
of unwounded and wounded samples to identify the significant spots
that were matched in all subjected gels. Among them, based on the gel
and spot quality, one gel was selected as the master gel for each con­
dition (UW/ W), against which all other respective gels were matched.
After matching, the differentially expressed protein spots in control
unwounded and wounded sample gels were identified along with their
fold change using Image Master 2D Platinum software (GE Healthcare)
according to the manufacturer’s instructions. Later, the protein spots
with fold changes >1.5 and p < 0.05 were considered as differentially
regulated proteins.
2.9. Peptide extraction and identification by MALDI-TOF-TOF
Differentially regulated protein spots were identified using MALDI-
TOF-TOF by following the procedure described by Kamaladevi and
Balamurugan 2017 with minor modifications [23]. Briefly, the protein
spots were excised manually from the gel using end-remove pipet tips
according to various diameter of spots. Later, the gel was destained
completely and lyophilised following dehydration with 30% acetonitrile
(ACN) and 100 mM ammonium bicarbonate (AmBiC) and subsequent
washes with 100% ACN. The protein spots were subjected to reduction
and alkylation by adding 10 mM DTT and 50 mM IAA, respectively.
Then the protein spots were dried and rehydrated in a protease solution
containing 2.5 ng/μl of Trypsin (Sigma) solution and incubated on ice
for 45 min. The mixture was incubated at 37 ◦ C for trypsinization. After
16 h of enzymatic digestion, the peptides were extracted by spin down
after addition of extraction buffer (50% ACN and 0.1% formic acid in 50
mM AmBiC). The extracts were vacuum dried at 45 ◦ C and suspended in
5 μl MS grade water and desalted using Zip TipC 18 pipet tips (Milli­
pore). About 1 μl of a- Cyano-4-hydroxycinnamic acid was spotted with
equal volume of desalted samples and allowed to dry for 4 h. The
MALDI-TOF/TOF analysis was performed using MALDI-TOF/TOF
3
Journal of Proteomics 240 (2021) 104222
analyser (AXIMA Performance, SHIMADZU BIOTECH) in a positive
reflectron ion mode. Subsequently, the proteins were identified through
peptide mass fingerprint (PMF) using MS Fit of ProteinProspector v 6.2.1
Tool.
2.10. LC-MS/MS analysis
Protocol described by Mir and Balamurugan with slight modifica­
tions was followed for LC-MS/MS analysis [24]. Briefly, 200 μg of pro­
tein (per sample) in 100 μl was subjected to incubation in dark for 1 h at
room temperature following addition of freshly prepared 200 mM DTT.
Further, 20 μl of 200 mM IAA was added and incubated at 56 ◦ C for 1 h.
Subsequently, the sample was subjected to in-solution trypsin digestion
using trypsin prepared in 55 mM acetic acid (1,50 ratio) following
addition of 200 mM DTT. Then the samples were incubated at 37 ◦ C for
16 h and proceeded for LC-MS/MS analysis (Agilent 6545 ESI-LC/qToF-
MS/MS) where the trypsin digested peptides were subjected to LC-MS/
MS analysis by employing liquid chromatography using Agilent 6545
ESI-LC/qToF-MS/MS and operated/controlled by Mass Hunter Work­
station software (version B.08.00) and the post processing of data was
performed using Spectrum Mill software (version B.06.00). Initially,
fractionation of peptides was performed using 1st Reversed Phase (RP)
column at high pH (pH 8.0) which is the first-dimension separation,
subsequently subjected to 2nd RP column at a low pH (pH 2.0) which is
the second dimension. In connection to the 2D separation, peptides from
the nano-LC column were subjected to MS analysis. The LC-MS/MS raw
data from each sample and each replicate were subjected to Spectrum
Mill software (version B.06.00) for the protein identification. The
C. elegans protein sequences were retrieved from NCBI which was used
for protein identification and the FDR (False Discovery Rate) of proteins
was set to 4%. Meanwhile, the post processing of LC-MS/MS raw data
was planned in such a way that the resulting protein must have at least
one fragment ion match and one peptide match. The taxonomy was set
as C. elegans and product mass tolerance was set to 1200 mDa. Later, the
total intensities of the spectra in triplicates of unwounded and wounded
samples were compared to calculate the fold change.
2.11. Protein data analysis
Interaction data of the identified/ interested proteins were acquired
using the Search tool for the retrieval of interacting genes (STRING)
online tool (Version 11.0) with a medium confidence score of 0.400
[25]. Major pathway(s) regulated from the identified proteins gene
enrichment of identified proteins was assessed using the KEGG Pathway
analysis provided by STRING. To identify the common proteins in both
unwounded and wounded samples, proteins identified from LC-MS/MS
analysis was subjected to Venny 2.1 online tool. Significant proteins
from common list of proteins were identified through volcano plot
analysis using GraphPad Prism version 8.4.3 [26].
2.12. Protein-protein interaction (PPI) network construction and module
analysis
PPI network of interested protein players were constructed using
STRING with medium confidence score 0.400 and fed into the visualized
by means of Cytoscape software version 3.8.0, written in Java [27].
Network was further analysed using network analyser plug-in in Cyto­
scape software and degree centrality of the nodes were obtained where
nodes with higher degrees were taken as key nodes. Additionally, the
significant module(s) were explored using molecular complex detection
(MCODE) plug-in in Cytoscape software [28,29]. The advanced options
set as degree cut off = 2, K-Core = 2, and Node Score Cut off = 0.2 [30].
Additional data of identified proteins were added to the PPI network
using Omics Visualizer plug-in in Cytoscape software [31].
M. Pooranachithra et al. Journal of Proteomics 240 (2021) 104222

Fig. 1. Glass wool mediated wounding of C. elegans. (A) Microscopic imaging of injured worms. Depth of the injury was increased from W1 to W5; (B) Assessment
of success rate of injury. In figure, Bar charts indicate injured worms, square dots indicate uninjured worms and line graph indicates disintegrated worms during the
respective injured worms; (C) Recovery assessment after injury. W1 and W2 display recovery of injury while W3-W5 display decaying of worms i.e. no recovery.

4
M. Pooranachithra et al. Journal of Proteomics 240 (2021) 104222

Fig. 2. 2-D GE of proteins isolated from unwounded and wounded worms. (A) Assessment of integrity of isolated proteins by 1-D SDS PAGE; (B) 2-D gel of
unwounded sample; (C) 2-D gel of wounded sample; (D) Total proteins identified from 2-D GE marked on C. elegans according to their expression profile obtained
using WormBase. [Protein spots of the significantly differential regulated proteins identified from the study is marked in red colored circle in the gel pictures]. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2.13. Identification of hub players and Gene Ontology of identified plotted forratio of 340 nm/380 nm against time where changes in 340/
proteins 380 ratio represents [Ca2+]i [36].

Hub players among the identified proteins from the PPI network
were identified using cytoHubba plugin in Cytoscape software [32,33].
Gene ontology, networks of the pathways (P < 0.05) and pathway-
associated genes from the identified proteins were analysed by the
Cytoscape plugin ClueGO [34]. Advanced term was set with minimal
restricted options which included one-ten GO tree terminals, minimum
one cluster and Kappa score 0.1 using C. elegans marker list containing
6239 nodes from ClueGO repository. For functional annotation of nodes,
gene specific interaction networks were performed using the Cytoscape
plugin, BiNGO [35].
2.14. Analysis of calcium signaling
To analyse calcium signaling, worms (~2000 numbers) were loaded
with 5 μM Fura-2 AM by incubating at 20 ◦ C for 20 mins. Fluorescence
was measured for Fura free/ saturated state with excitation and emission
at 340 nm and 510 nm & 380 nm and 510 nm, respectively for
consecutive 10 min with 12 S break for 0, 3, 5, 21 and 24 h intervals
using multi-label reader (Spectramax M3, USA). Then the graph was
2.15. Analysis of mitochondrial fission in assisting wound repair through
the expression of PINK-1 using transgenic worm
pink-1 was uncovered to function as a scout to monitor the damaged
mitochondria and also reported to control the mitochondria quality
through mitochondrial fission [37,38]. Based on that, BR4006 [pink-1p::
pink-1::GFP + myo-2p::mCherry + herring sperm DNA], PINK-1 trans­
genic worms were opted for the present study where worms were
washed and subjected to wounding process. Later the GFP quantified
using multi-label reader with excitation and emission at 395 nm and
509 nm.
2.16. Trypan blue (TryB) staining for assessment of wound repair
Membrane-impermeable dye, Trypan blue (TryB) was used as a
marker of wound repair where prevention of dye staining is considered
as repaired. Wounds that could be permeated with TryB indicated by the
blue colour around the wound site(s) suggested impairment of wound
repair. However, wounds with no or faint blue colour was considered as

5
M. Pooranachithra et al. Journal of Proteomics 240 (2021) 104222

Table 1
List of up-regulated proteins identified from 2-D gel electrophoresis.
S. Match UniProt ID Acc. No. Gene name Protein name % of % of MOWSE Fold p-Value
No. ID Match Coverage score change

1 137 CNP3_CAEEL O02039 cnp-3 Calcineurin-interacting protein 3 60 6.80% 1092 3.24132 0.0110811
2 641 VHL_CAEEL Q19213 vhl-1 von Hippel-Lindau tumor suppressor 100% 14.37% 32.3 2.37776 0.0121526
homolog
3 54 YOTB_CAEEL P34657 ZK632.12 Uncharacterized protein ZK632.12 16% 13.50% 779 2.334 0.0233719
4 379 NAS14_CAEEL Q19269 nas-14 Zinc metalloproteinase nas-14 54% 9.10% 1135 2.21714 0.0176188
5 285 NHR61_CAEEL O62389 nhr-61 Nuclear hormone receptor family 80% 6.30% 58.7 2.21529 0.0475598
member nhr-61
6 68 SYNJ_CAEEL G5ECL2 unc-26 Synaptojanin 100% 7.33% 839 2.17352 0.0291487
7 240 ACH5_CAEEL Q23022 unc-38 Acetylcholine receptor subunit alpha- 80% 4.90% 100 2.10653 0.0312654
type unc-38
8 934 YPI7_CAEEL Q18262 C27F2.7 Uncharacterized F-box protein C27F2.7 100% 4.17% 40.7 2.0888 0.0380669
9 926 FAT3_CAEEL Q23221 fat-3 Delta(6)-fatty-acid desaturase fat-3 80% 5.90% 46 2.08157 0.0308132
10 850 KPC2_CAEEL P90980 pkc-2 Protein kinase C-like 2 57% 3.8% 61.2 2.07882 0.0497419
11 29 YKAA_CAEEL P34261 B0303.11 Uncharacterized amino-acid permease 45% 3.10% 222 2.02758 0.022752
B0303.11
12 326 SET23_CAEEL Q95Y12 set-23 Probable histone-lysine N- 45% 14.80% 253 1.91604 0.0418761
methyltransferase set-23
13 796 YXT2_CAEEL Q18078 C18B2.2 Uncharacterized protein C18B2.2 66% 7.00% 172 1.86378 0.036121
14 399 NH103_CAEEL O16359 nhr-103 Nuclear hormone receptor family 80% 7.10% 59.9 1.86306 0.030579
member nhr-103
15 434 PK1_CAEEL Q17850 pak-1 Serine/threonine-protein kinase pak-1 100% 4.55% 77.8 1.85802 0.010126
16 407 GCY9_CAEEL E7EAU8 gcy-9 Receptor-type guanylate cyclase gcy-9 80% 1.7% 46.6 1.85554 0.025935
17 663 UNC86_CAEEL P13528 unc-86 Transcription factor unc-86 57% 10.10% 155 1.83406 0.012765
18 86 CRN4_CAEEL Q10905 crn-4 Cell death-related nuclease 4 55% 11.40% 270 1.75404 0.044958
19 18 CDKAL_CAEEL Q8MXQ7 Y92H12BL.1 Threonylcarbamoyladenosine tRNA 100% 6.12% 437 1.71302 0.028546
methylthiotransferase
20 1092 OLA1_CAEEL P91917 ola-1 Obg-like ATPase 1 50% 7.1% 143 1.68922 0.015033
21 38 EIF3L_CAEEL Q95QW0 eif-3 Eukaryotic translation initiation factor 3 50% 11.50% 364 1.62529 0.018546
subunit L
22 346 HCDH2_CAEEL P41938 B0272.3 Probable 3-hydroxyacyl-CoA 40% 12.60% 204 1.61835 0.032169
dehydrogenase B0272.3

repaired [39]. In the present study, wounded worms with unwounded
control (wild type and BR4006 transgenic worm) were stained with
0.01% final concentration of TryB for 15 min at 20 ◦ C in dark. After
incubation, worms were washed thrice with M9, imaged in groups for
the penetration of TryB using fluorescent microscope.
2.17. Statistical analysis
Statistical significance of the data was analysed through One-way
ANOVA (α = 0.05) followed by the multiple comparisons test, Tukey
statistical hypothesis testing using GraphPad Prism version 8.4.3 [40].
3. Results
3.1. W1 displayed recovery after glass wool mediated injury
Pertaining to the requirement of umpteen numbers on wounded
worms, glass wool mediated wounding protocol was opted for the study.
To examine the incidence of recovery/ repair process after glass wool
mediated injury, worms were injured by vortex for different time points
(1–10 min) with truncated glass wool pieces. This included five different
groups W1 (1 min), W2 (3 min), W3 (5 min), W4 (7 min), and W5 (10
min). As shown in Fig. 1A all five groups of worms displayed the

Table 2
List of down-regulated proteins identified from 2-D gel electrophoresis.
S. Match UniProt ID Acc. No. Gene name Protein name % of % of MOWSE Fold p-Value
No. ID Match Coverage score change

1 164 CL161_CAEEL P34472 clec-161 C-type lectin domain-containing protein 100% 5.22% 78.8 2.7068 0.0143433
161
2 143 DXO_CAEEL Q10660 dom-3 Decapping nuclease dom-3 80% 6.90% 138 2.68364 0.010892
3 867 NDC1_CAEEL Q8I4N3 npp-22 Nucleoporin ndc-1 66% 4.10% 27.9 2.12388 0.042366
4 753 SPDL1_CAEEL Q17695 spdl-1 Spindly-like protein spdl-1 71% 9.20% 355 2.10842 0.026423
5 331 MIG18_CAEEL O45348 mig-18 Abnormal cell migration protein 18 75% 12.30% 28.6 2.05731 0.072728
6 60 HCDH1_CAEEL P34439 F54C8.1 Probable 3-hydroxyacyl-CoA 75% 6.7% 68.7 1.97788 0.046031
dehydrogenase F54C8.1
7 464 CUL1_CAEEL Q17389 cul-1 Cullin-1 100% 7.95% 6.92 1.97561 0.032385
8 929 PRI2_CAEEL O02334 PRI2_CAEEL DNA primase large subunit 100% 4.97% 40.8 1.91584 0.017635
9 341 RSP2_CAEEL Q23120 rsp-2 Probable splicing factor,arginine/serine- 23% 11.70% 1001 1.90109 0.027722
rich 2
10 620 TXTP_CAEEL P34519 K11H3.3 Putative tricarboxylate transport protein, 100% 7.69% 58.4 1.88716 0.014585
mitochondrial
11 556 FABP5_CAEEL Q03568 lbp-5 Uncharacterized protein C38C10.3 100% 13.97% 59.5 1.83311 0.025193
12 826 MMSA_CAEEL P52713 alh-8 Probable methylmalonate-semialdehyde 100% 3.63% 35.2 1.67049 0.028945
dehydrogenase [acylating],
mitochondrial
13 234 GLS1_CAEEL Q8I4M5 gls-1 Germline survival defective-1 100% 4.94% 80.9 1.61853 0.018658

6
M. Pooranachithra et al. Journal of Proteomics 240 (2021) 104222

Fig. 3. PPI network analysis of proteins isolated from 2-D GE. (A) PPI network of upregulated protein;(B) GO of up regulated proteins; (C) PPI network of down
regulated protein; (D) GO of down regulated proteins. (Proteins identified from the study is indicated by blue colored nodes while their interacting partners are
colored in green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Molecular players involved in central biological events upon injury. (A) Acetylcholine transport; (B) Microtubule organization; (C) Phosphatidylinositol
signaling. (Molecular players that are involved in the event are colored in red). (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

7
M. Pooranachithra et al.
Fig. 5. Assessment of calcium signaling after injury. (A) [Ca2+]i at 0 h in unwounded and wounded worms; (B) [Ca2+]i at 3 h in unwounded and wounded worms;
(C) [Ca2+]i at 5 h in unwounded and wounded worms; (D) [Ca2+]i at 21 h in unwounded and wounded worms; (E) [Ca2+]i at 24 h in unwounded and
wounded worms.
incidence of injury where the depth of injury was varied based on the
time taken for vortex. However, W4 and W5 showed increase in numbers
(~20%) of disintegrated worms whereas W1 and W2 displayed less than
5% of disintegrated worms. In addition, W1 displayed less numbers of
uninjured worms compared to W2 (Fig. 1B) and all the injured worms
displayed locomotion defect except W1 and W2 (Data not shown). Re­
covery assessment of injured worms in NGM plates after 24 h showed
incidence of recovery/ repair in groups W1 and W2. While the other
groups W3, W4, W5 displayed no recovery but only the presence of
decaying dead worms at the spot where the injured worms were trans­
ferred (Fig. 1C). Based on these observations, W1 (hereafter indicated as
‘W’) was taken for further gel-based and gel-free proteomic analysis
including 2-D GE and LC-MS/MS, respectively.
3.2. 2-D GE analysis revealed the activation of calcium signaling
2-D GE was performed in triplicates for unwounded and wounded
samples (Suppl. Fig. S1) after confirming their quantity, quality and
integrity in 1-D SDS PAGE (Fig. 2A). In total, 35 significantly differential
regulated proteins were identified from comparison of 2-D gels of un­
wounded (Fig. 2B) and wounded (Fig. 2C) samples in which 22 (Table 1)
were upregulated and 13 (Table 2) were down regulated. All the iden­
tified proteins are marked in the Fig. 2D according to their expression
profiles. PPI network of up and down regulated proteins were visualized
in Cytoscape network panel as shown in Fig. 3A where the up regulated
proteins that are identified from the 2-D GE analysis are displayed in
blue. Rest of the nodes in green colour in the network are the interacting
partners of the identified up regulated proteins. Analysis of highly
interconnected regions/ clusters in the network using MCODE plugin
from Cytoscape resulted with three clusters from the PPI network of up
8
Journal of Proteomics 240 (2021) 104222
regulated proteins which included endocytosis, phagosomes and tryp­
tophan metabolism. Among that, tryptophan metabolism which is
pointed in the Fig. 3A by eclipse was expected to be crucial as it is linked
with the serotonin synthesis. Similarly, MCODE analysis of PPI network
of down regulated proteins resulted with two clusters in which one
cluster contributed to DNA replication/ base excision repair and other
cluster for ubiquitin mediated proteolysis as indicated in Fig. 3C by
eclipse. In addition, GO analysis of identified up regulated proteins
resulted with number of regulatory events as displayed in Fig. 3B
including acetylcholine transport and phosphatidylinositol binding.
Likewise, GO of down regulated proteins included microtubule orga­
nizing center organization and mechanosensory behavior (Fig. 3D).
Notably, fat-3, unc-26, pkc-2 and gcy-9 were involved in acetylcholine
transport (Fig. 4A) whereas spdl-1, cul-1, pkc-2, fat-3 were involved in
microtubule organizing center organization (Fig. 4B) and ZK632.12 was
involved in phosphatidylinositol binding (Fig. 4C). To further verify the
insights obtained from PPI network analysis of up regulated/ down
regulated proteins, PPI network of total proteins identified from 2-D GE
was analysed. Results also justified the involvement of serotonin/
tryptophan secretion, positive regulation of neuronal action potential,
tryptophan metabolism, phosphatidylinositol signaling and calcium
signaling in response to injury (Suppl. Fig. S2).
Identification of phosphatidylinositol binding from the identified
players indicated the activation of calcium signaling after glass wool
mediated injury. Additionally, the status of phosphatidylinositol binding
after injury was assessed by analyzing the gene responsible for inositol
try phosphate receptor(itr-1), a downstream player of phosphatidyli­
nositol binding using qPCR analysis. Results indicated upregulation of
the same at the initial hours and downregulated at the later hours post
injury (Suppl. Fig. S3). Moreover, the involvement of the calcium
M. Pooranachithra et al. Journal of Proteomics 240 (2021) 104222

Fig. 6. Analysis of proteins obtained from LC-MS/MS. (A) Venn diagram analysis of proteins identified from unwounded and wounded samples at 0 and 24 h; (B)
Volcano Plot analysis of proteins identified from unwounded and wounded samples at 0 and 24 h where proteins with p-Value ≤0.05 appears at the top of the plot
separated by the blue line in the figure and indicated, red in colour; (C) PPI network analysis of common proteins from unwounded and wounded conditions at 0 h;
(D) PPI network analysis of common proteins from unwounded and wounded conditions at 24 h (Up regulated, down regulated, other common proteins are colored in
green, red and Turquoise blue respectively). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)

signaling after glass wool mediated injury, was confirmed by staining
using Fura 2-AM. [Ca2+]i measured after injury at different time periods
including 0, 3, 5, 21 and 24 h by calculating 340/380 ratio is displayed
respectively in Fig. 5A–E. Calcium signaling immediately after injury
was higher than the control unwounded worms (Fig. 5A) which was
reduced during the incubation period and equal to the [Ca2+]i in un­
wounded worms at 24 h (Fig. 5E).
3.3. LC-MS/MS analysis revealed induction of actomyosin organization
and mitochondrial fission
In total, 981 and 895 proteins were identified in unwounded and
wounded samples, respectively, at 0 h. In addition, 918 and 909 proteins
were identified in unwounded and wounded samples, respectively, at
24 h. Among this 210 (Suppl. Table S2) and 225 (Suppl. Table S3) were
found to be common for both unwounded and wounded conditions,
respectively (Fig. 6A). Further subjugation of the common protein to
volcano plotting resulted with 13 (Table 3) and 10 (Table 4) significant
proteins at 0 and 24 h, respectively (Fig. 6B). PPI network of the sig­
nificant proteins identified at 0 and 24 h was constructed (Suppl. Fig.­
S4A& C) and subjected to ClueGO analysis which indicated cortical actin
cytoskeleton organization, protein phosphatase binding, proton trans­
porting ATP synthase activity at 0 h (Suppl. Fig. S4B); skeletal muscle
myosin thick filament assembly, actin filament depolymerization at 24 h
(Suppl. Fig. S4D). Interestingly, both reactive oxygen species
biosynthetic process and negative regulation of the same was found to be
regulated by the significant proteins identified at 24 h (Suppl. Fig. S4D).
PPI network of the common proteins identified at 0 and 24 h is
displayed in Fig. 6C&D respectively, where the upregulated protein
nodes are colored in green while the down regulated protein nodes are
colored in red. Further analysis of biological processes through ClueGO
indicated the incidence of positive regulation of actin nucleation at 0 h
(Fig. 7A) and RHO protein signal transduction, myosin filament as­
sembly at 24 h (Fig. 7B). Besides, a closer look into the biological process
of common proteins identified at 24 h specified the involvement of
phosphatidylinositol dephosphorylation, myosin filament assembly,
actomyosin structure organization and most importantly positive regu­
lation of mitochondrial fission (Fig. 7C). In addition, pink-1 was found to
be responsible for the positive regulation of mitochondrial fission
(Fig. 7D) and unc-87, unc122, unc-54, unc-15, unc-82, mks-3, sas-4, dys-1,
gad-1, act-1, mlc-1, tbb-1, tln-1, cpna-1,osm-1, acbp-1, atp-2, C34D4.1
were found to be involved in actomyosin structure organization
(Fig. 7E). In addition, pink-1 was found to directly interact with ubq-1,
tag-173 and Y40C5A.1 as depicted in Suppl. Fig. S5A with all other sub
interaction players. Further, BiNGO analysis of the pink-1 interaction
network inferred ubiquitin dependent protein catabolic process (Suppl.
Fig. S5B) and positive regulation of growth (Suppl. Fig. S5C). Further,
qPCR analysis of pink-1 gene at various time points post injury (0, 4, 8,
12, 24 h) clearly indicated the upregulation of pink-1 gene at the initial
hours post injury (0, 4 h) and started to reduce from 8 h till 24 h post

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M. Pooranachithra et al. Journal of Proteomics 240 (2021) 104222

Table 3 incidence myosin filament assembly, actin cytoskeleton, actomyosin

Significant (p < 0.05) differentially regulated proteins (by comparing un­ complex at 0 h (Suppl. Fig. S6A) and contractile fiber, muscle myosin
wounded and wounded group) immediately after injury using LCMS/MS complex at 24 h (Suppl. Fig. S6B). Further, molecular function analysis
analysis. showed Ras GTPase binding at 0 h (Suppl. Fig. S6C) and actin and
S. UniProt Protein Name Gene Fold p-Value cytoskeletal protein binding at 24 h (Suppl. Fig. S6D).
No. ID Name change

1 G5EBF3 Protein Up-regulated pud-2.1 0.578947 0.047421 3.4. Pathway analysis exposed calcium and Hippo signaling upon injury
in daf-2
2 G8JY38 Deleted. 0.5 0.047421
–
Initially, proteins identified from unwounded, wounded worms at
3 Q18688 Heat shock protein 90 daf-21 0.2 0.047421
4 P09446 Heat shock 70 kDa hsp-1, 0.4375 0.033472 0 and 24 h were subjected to pathways enrichment analysis via STRING
protein A hsp70a database. Results indicated that inositol phosphate metabolism and
5 P53013 Elongation factor 1- eft-3, eft- 0.492063 0.029918 phosphatidylinositol signaling that mediate calcium signaling upon
alpha (EF-1-alpha) 4 wounded condition at 24 h. However, only ribosome was enriched from
6 Q9BKU5 Ribosomal_L18e/L15P Y37E3.8 0.285714 0.02411
unwounded condition at 0 and 24 h, wounded conditions at 0 h (Suppl.
domain-containing
protein Fig. S7). Additionally, PPI network analysis of the significant differential
7 P0DM41 Actin-1 act-1 0.46875 0.02227 regulated proteins identified from the study indicated enrichment of
8 Q9XXK1 ATP synthase subunit atp-1 0.423077 0.022003 calcium signaling pathway, oxidative phosphorylation, MAPK signaling
alpha, mitochondrial
pathway etc., at 0 h and cysteine methionine metabolism, ribosome and
(atp-1)
9 J7S164 Histone H2A his-57 0.733333 0.01613
phagosome at 24 h (Suppl. Fig. S8). Moreover, proteins identified from
10 O01805 Acyl-CoA-binding acbp-1 0.333333 0.01613 unwounded and wounded samples at 0 h were subjected to KEGG
protein homolog 1 mapper search pathway online application which indicated the differ­
(ACBP-1) ence in induction of calcium signaling (Suppl. Fig. S9). Notably in un­
11 P49041 40S ribosomal protein rps-5 0.142857 0.013236
wounded condition, calcium signaling was induced by the growth
S5
12 O45865 Adenine Nucleotide ant-1.1 0.227273 0.01047 factors that in turn contributed for the contraction (Suppl. Fig. S9A).
Translocator However, in wounded condition, calcium signaling was induced by the
13 P46561 ATP synthase subunit atp-2 0.0625 0.006074 neurotransmitters that in turn contributed to the proliferation (Suppl.
beta, mitochondrial
Fig. S9B). For further insights into pathways involved, common proteins
from 0 and 24 h were subjected to ClueGO analysis. The results indicated
the activation of calcium signaling and Notch signaling at 0 h (Fig. 9A)
while Hippo signaling pathway at 24 h (Fig. 9B).
injury (Fig. 8A). Furthermore, survival analysis after wounding in
BR4006 transgenic model showed difference in survival after wounding 4. Discussion
compared to wild type at 24 h (Fig. 8B). In addition, TryB staining of
worms at 24 h also indicated improved recovery of wounded worms in Though, C. elegans is being explored as a wound model for more than
BR4006 compared to wild type (Fig. 8C). Quantification of Pink-1::GFP a decade, global proteomic changes are not looked at yet upon injury
after wounding of BR4006 indicated comparatively higher intensity in and following repair. In this regard, the present study was carried out in
wounded conditions at 0, 12 h and reduced at 24 h (Fig. 8D). Addi­ C. elegans to uncover new insights from wound repair through global
tionally, analysis of cellular processes through ClueGO indicated the proteomic analysis. For this purpose, 2-D GE and LC-MS/MS analysis
was carried out in unwounded and wounded samples and the PPI
network constructed from the identified proteins included 2683 non
redundant nodes which is actually 1/10thof the total nodes present in
Table 4 C. elegans whole PPI network available in the database (Suppl. Fig. S10).
Significant (p < 0.05) differentially regulated proteins (by comparing un­ This is an added value of the present study which resulted with fruitful
wounded and wounded group) ay 24 h using LCMS/MS analysis. insights into crucial regulatory players and pathways that are regulated
S. UniProt ID Protein Name Gene Fold p-Value
during wound repair.
No. Name change Remarkably, tryptophan metabolism was identified from the PPI
network analysis of up regulated proteins whereas GO analysis of the
1 P18948 Vitellogenin-6 vit-6 0.511628 0.048502
2 P27604 Adenosyl ahcy-1 0.2 0.047421 same indicated acetylcholine transport and phosphatidylinositol bind­
homocysteinase ing. Interestingly, identification of tryptophan metabolism indicates the
(AdoHcyase) active role of serotonin neurotransmitter in wound repair as tryptophan
3 G5EET8 PUD1_2 domain- pud- 0.416667 0.024896 is the sole precursor of serotonin [41]. In addition, acetylcholine
containing protein 1.2
4 Q95Y04 40S ribosomal rps-28 0.3 0.024896
transport identification also indicates the other possible action of
protein S28 acetylcholine neurotransmitter in wound repair. Previous studies in
5 P02566 Myosin-4 (Myosin unc- 0.324324 0.0189 mammals also support the active role of the above-mentioned neuro­
heavy chain B) 54 transmitters in facilitating the process of repair [42,43]. In addition,
6 G5EBF3 Protein Up-regulated pud- 0.48 0.014721
finding of phosphatidylinositol binding in the earlier study denotes the
in daf-2 2.1
7 O17921 Tubulin beta chain tbb-1 0.125 0.007763 active involvement of calcium signaling [44,45] which is well corrob­
8 A0A0K3AQS9 Uncharacterized lev-11 0.428571 0.002278 orated in the present study using Fura-2 AM.
protein Additionally, PPI network analysis of down regulated proteins sug­
9 O02640 Probable malate mdh-2 0.090909 0.002111 gested that the ubiquitin mediated proteolysis was compromised upon
dehydrogenase,
mitochondrial
injury. This is substantiated with a recent finding where ubiquitin
10 P10567 Paramyosin unc- 0.076923 0.001058 mediated proteolysis of molecular chaperons related proteins were re­
(Uncoordinated 15 ported to negatively regulate wound repair [46]. Microtubule organi­
protein 15) zation identified from the GO of down regulated proteins indicated the
activation of fat-3 in response to pkc-2 specified the link for fat meta­
bolism and calcium signaling. A previous study is in corroboration with

10
M. Pooranachithra et al.
Fig. 7. GO analysis of proteins identified using LCMS/MS. (A) GO of common proteins identified at 0 h; (B) GO of common proteins identified at 24 h; (C) Central
biological events regulated from common proteins identified at 24 h; (D) Molecular players involved in mitochondrial fission; (E) Molecular players involved in
actomyosin structure organization.
the above fact where link between microtubule regulators and fat
metabolism was discussed [47]. Based on the PPI network, we also hy­
pothesize that the fat metabolism in relation with microtubule organi­
zation could be in downstream of calcium signaling. However,
experimental evidence is required in support of the above-mentioned
phenomena.
Further, it was predicted that protein phosphatase binding, ATP
synthase activity facilitate the actomyosin cable formation and
following repair in accordance with the previous finding where protein
kinases were reported to regulate myofilaments [48]. Identification of
actin nucleation from PPI network analysis of common regulators at 0 h
indicates the initiation of actin polymerisation after injury while iden­
tification of RHO protein signal transduction and myosin filament as­
sembly at 24 h suggested the repair through actomyosin complex
formation via RHO protein signaling. This is in authentication with the
earlier finding where the role of RHO proteins in wound repair are
highlighted [49]. Additionally, identification of phosphatidylinositol
dephosphorylation at 24 h analysis implied the regulation of calcium
signaling to normal which was also corresponded with the experimental
[Ca2+]i in our study.
Notably, positive regulation of mitochondrial fission was identified
where pink-1 was involved. This is in corroboration with the earlier
findings where pink-1 was reported to control mitochondrial fission and
motility [50,37]. Based on that, it was suspected that pink-1 could have
mediated positive regulation of mitochondrial fission to promote wound
repair. Moreover, this finding is in accordance with the recent report
11
Journal of Proteomics 240 (2021) 104222
where the crucial role of mitochondrial fission in F-actin dynamics,
accelerated wound closure and localised signaling downstream of cal­
cium signaling for the repair after injury was described [10–12]. In
addition, over expression of pink-1 was found in our study which is also
in support of the earlier finding where overexpression of PINK1 was
reported to mediate mitochondrial fission [51]. Moreover, it was also
reported that mitochondrial fission promotes ubiquitin mediated pro­
teolysis to facilitate the increased uptake of mitochondrial Ca2+ [52].
This is in support of our finding where pink-1 was identified to be
resulted with ubiquitin mediated proteolysis and growth. Additionally,
activation of Hippo signaling was identified in our study wherein
another recent report attested the crucial role of Hippo signaling in
wound repair and regenerative medicine [53,54]. Besides, the impact
and exact role of Hippo signaling in wound repair requires further
research. Further, PPI network analysis of significantly differential
proteins identified from LCMS/MS analyses indicated MAPK signaling
which in turn is expected to culminate in innate immune response after
injury. Furthermore, number of kinases including Serine/threonine-
protein kinase, Tyrosine-protein kinase and Mitogen-activated protein
kinase kinase kinase were identified during wounded condition where
kinase activities were reported as essential for its function in innate
immunity [55]. These are in support of the recent finding where active
role of innate immunity and its correlation with the microtubule dy­
namics was reported during repair [56].
Overall, the study investigated the molecular players involved in
wound healing through 2-D GE and LCMS/MS analyses where different
M. Pooranachithra et al. Journal of Proteomics 240 (2021) 104222

Fig. 8. pink-1 promotes wound repair in C. elegans. (A) quantification of pink-1 transcript using qPCR at various time points post injury in wild type C. elegans (B)
Survival analysis after wounding in wildtype and BR4006 transgenic model (C) TryB staining mediated assessment of repair in wildtype and BR4006 transgenic
model (D) Quantification of GFP in BR4006 after injury. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)

12
M. Pooranachithra et al. Journal of Proteomics 240 (2021) 104222

Fig. 9. Pathways enriched in proteins identified using LCMS/MS. (A) ClueGO result of enriched pathway from common proteins identified at 0 h; (B) ClueGO
result of enriched pathway from common proteins identified at 24 h [In the figure, size of the nodes reflects the enrichment significance of the terms where functional
groups represented by their most significant (leading) term are visualized in the network with random colors providing an insightful view of their interrelations].

number of regulatory proteins were identified. However, the crucial
biological events activated in response to injury such as phosphatidyli­
nositol signaling involved in calcium signaling was found from both the
analytical techniques where LCMS/MS analyses provided more insights
into the molecular players and the crucial biological events that are
activated in response to injury. Based on our proteomic data using
C. elegans wound model and the referred earlier reports we outlined the
possible regulations of crucial players along the pathways involved as
shown in Fig. 10. Overall, we propose that injury triggers calcium
through phosphatidylinositol signaling which in turn activates neuro­
transmitters, actin nucleation, actomyosin complex formation, mito­
chondrial fission, ubiquitin mediated proteolysis and hippo signaling to
repair the injury in C. elegans. However, validation of the pathways in
response to injury is required for further confirmation.

13
M. Pooranachithra et al. Journal of Proteomics 240 (2021) 104222

Fig. 10. Overall mechanism of molecular players and pathways regulated upon injury in C. elegans. Wounding of C. elegans initiates calcium signals and
neurotransmitters. This in turn activates actin nucleation, actomyosin organization, mitochondrial fission, ubiquitin mediated proteolysis, Notch signaling and Hippo
signaling in response to injury for the accelerated repair. In figure, the molecular layers identified from the study that are predicted to be involved in the central
repair events are colored in pink. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Authors’ information Ethics approval

MP, is a Ph. D research scholar, engaged in exploring the interesting
facts regarding wound healing using model nematode C. elegans and
awarded Augmenting Writing Skills for Articulating Research (AWSAR)
2018 for popular science story entitled “Wound Research: Insights from
a Mighty Miniature Model, Caenorhabditis elegans” by Department of
Science and Technology (DST), Government of India. KB, is a Professor
in the Department of Biotechnology, majorly interested in “Host
pathogen-interactions studies using C. elegans as a model organism by
functional genomics, proteomics and metabolomics approach. KV is
Chief Scientist, BJP is Senior Principal Scientist, at ITC Limited. CSK is
master’s student at Department of Biotechnology, Alagappa University.
VR is the Director of NIPER, Kolkata.
Credit author statement
M.P., K⋅B and J.P⋅B conceived the research; M.P., C.S.K. conducted
the 2-D GE analysis; M.P conducted all the experiments, analysed the
data and wrote the manuscript; M.P. and V. R. reviewed the LC-MS/MS
analysis; M.P., B.J.P., K⋅V and K⋅B: reviewed the experimental design,
manuscript and had primary responsibility for the final content of the
manuscript. All authors read and approved the final manuscript.
Funding
Not applicable.
This article does not contain any studies with human or animal
subjects performed by the any of the authors.
Declaration of Competing Interest
None.
Acknowledgments
Authors thank the Caenorhabditis Genetics Center, which is funded by
the National Institute of Health, National Centre for Research Resources
for providing the nematode strains. Authors acknowledge the ITC-LSTC,
Bangalore for the financial assistance in the form of project fellowship.
Technical assistance provided by Mr. B. Balasubramaniam, Research
Scholar, Department of Biotechnology, Alagappa University and Gajb­
hiye Rahul, Research Scholar, National Institute of Pharmaceutical Ed­
ucation and Research in performing LC-MS/MS analysis is
acknowledged. Authors also acknowledge the University Science
Instrumentation Centre (USIC), Alagappa University. Instrumentation
Facility provided by Department of Science and Technology (DST),
Government of India through DST PURSE [Grant No. SR/S9Z-415
/2010/42(G)], DST FIST [Grant No. SR/FST/LSI-639/2015(C)], UGC
through SAP-DRS1 [Grant No. F.5-1/2018/DRS-II (SAPII)] and RUSA
2.0 [Grant No. F. 24- 51/2014-U, Policy (TN Multi-Gen), Dept of Edn,
GOI], LC-MS/MS instrumentation facility from NIPER, Kolkata are
thankfully acknowledged. “The mass spectrometry proteomics data

14
M. Pooranachithra et al.
have been deposited to the ProteomeXchange Consortium via the PRIDE
partner repository with the dataset identifiers PXD024629/
PXD024744”.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jprot.2021.104222.
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