Paxillin interactome identified by SILAC and label-free approaches coupled to TurboID sheds light on the compositions of focal adhesions in mouse embryonic stem cells.

Self-renewal and differentiation of mouse embryonic stem cells (mESCs) are greatly affected by the extracellular matrix (ECM) environment; the composition and stiffness of which are sensed by the cells via integrin-associated focal adhesions (FAs) which link the cells to the ECM. Although FAs have been studied extensively in differentiated cells, their composition and function in mESCs are not as well elucidated. To gain more detailed knowledge of the molecular compositions of FAs in mESCs, we adopted the proximity-dependent biotinylation (BioID) proteomics approach. Paxillin, a known FA protein (FAP), is fused to the promiscuous biotin ligase TurboID as bait. We employed both SILAC- and label-free (LF)-based quantitative proteomics to strengthen as well as complement individual approach. The mass spectrometry data derived from SILAC and LF identified 38 and 443 proteins, respectively, with 35 overlapping candidates. Fifteen of these shared proteins are known FAPs based on literature-curated adhesome and 7 others are among the reported "meta-adhesome", suggesting the components of FAs are largely conserved between mESCs and differentiated cells. Furthermore, the LF data set contained an additional 18 literature-curated FAPs. Notably, the overlapped proteomics data failed to detect LIM-domain proteins such as zyxin family proteins, which suggests that FAs in mESCs are less mature than differentiated cells. Using the LF approach, we are able to identify PDLIM7, a LIM-domain protein, as a FAP in mESCs. This study illustrates the effectiveness of TurboID in mESCs. Importantly, we found that application of both SILAC and LF methods in combination allowed us to analyze the TurboID proteomics data in an unbiased, stringent and yet comprehensive manner.

The scaffold RhoGAP protein ARHGAP8/BPGAP1 synchronizes Rac and Rho signaling to facilitate cell migration.

Rho GTPases regulate cell morphogenesis and motility under the tight control of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). However, the underlying mechanism(s) that coordinate their spatiotemporal activities, whether separately or together, remain unclear. We show that a prometastatic RhoGAP, ARHGAP8/BPGAP1, binds to inactive Rac1 and localizes to lamellipodia. BPGAP1 recruits the RacGEF Vav1 under epidermal growth factor (EGF) stimulation and activates Rac1, leading to polarized cell motility, spreading, invadopodium formation, and cell extravasation and promotes cancer cell migration. Importantly, BPGAP1 down-regulates local RhoA activity, which influences Rac1 binding to BPGAP1 and its subsequent activation by Vav1. Our results highlight the importance of BPGAP1 in recruiting Vav1 and Rac1 to promote Rac1 activation for cell motility. BPGAP1 also serves to control the timing of Rac1 activation with RhoA inactivation via its RhoGAP activity. BPGAP1, therefore, acts as a dual-function scaffold that recruits Vav1 to activate Rac1 while inactivating RhoA to synchronize both Rho and Rac signaling in cell motility. As epidermal growth factor receptor (EGFR), Vav1, RhoA, Rac1, and BPGAP1 are all associated with cancer metastasis, BPGAP1 could provide a crucial checkpoint for the EGFR-BPGAP1-Vav1-Rac1-RhoA signaling axis for cancer intervention.

Zyxin regulates embryonic stem cell fate by modulating mechanical and biochemical signaling interface.

Biochemical signaling and mechano-transduction are both critical in regulating stem cell fate. How crosstalk between mechanical and biochemical cues influences embryonic development, however, is not extensively investigated. Using a comparative study of focal adhesion constituents between mouse embryonic stem cell (mESC) and their differentiated counterparts, we find while zyxin is lowly expressed in mESCs, its levels increase dramatically during early differentiation. Interestingly, overexpression of zyxin in mESCs suppresses Oct4 and Nanog. Using an integrative biochemical and biophysical approach, we demonstrate involvement of zyxin in regulating pluripotency through actin stress fibres and focal adhesions which are known to modulate cellular traction stress and facilitate substrate rigidity-sensing. YAP signaling is identified as an important biochemical effector of zyxin-induced mechanotransduction. These results provide insights into the role of zyxin in the integration of mechanical and biochemical cues for the regulation of embryonic stem cell fate.

Transcriptomics indicate nuclear division and cell adhesion not recapitulated in MCF7 and MCF10A compared to luminal A breast tumours.

Breast cancer (BC) cell lines are useful experimental models to understand cancer biology. Yet, their relevance to modelling cancer remains unclear. To better understand the tumour-modelling efficacy of cell lines, we performed RNA-seq analyses on a combined dataset of 2D and 3D cultures of tumourigenic MCF7 and non-tumourigenic MCF10A. To our knowledge, this was the first RNA-seq dataset comprising of 2D and 3D cultures of MCF7 and MCF10A within the same experiment, which facilitates the elucidation of differences between MCF7 and MCF10A across culture types. We compared the genes and gene sets distinguishing MCF7 from MCF10A against separate RNA-seq analyses of clinical luminal A (LumA) and normal samples from the TCGA-BRCA dataset. Among the 1031 cancer-related genes distinguishing LumA from normal samples, only 5.1% and 15.7% of these genes also distinguished MCF7 from MCF10A in 2D and 3D cultures respectively, suggesting that different genes drive cancer-related differences in cell lines compared to clinical BC. Unlike LumA tumours which showed increased nuclear division-related gene expression compared to normal tissue, nuclear division-related gene expression in MCF7 was similar to MCF10A. Moreover, although LumA tumours had similar cell adhesion-related gene expression compared to normal tissues, MCF7 showed reduced cell adhesion-related gene expression compared to MCF10A. These findings suggest that MCF7 and MCF10A cell lines were limited in their ability to model cancer-related processes in clinical LumA tumours.

Zyxin Is Involved in Fibroblast Rigidity Sensing and Durotaxis.

Focal adhesions (FAs) are specialized structures that enable cells to sense their extracellular matrix rigidity and transmit these signals to the interior of the cells, bringing about actin cytoskeleton reorganization, FA maturation, and cell migration. It is known that cells migrate towards regions of higher substrate rigidity, a phenomenon known as durotaxis. However, the underlying molecular mechanism of durotaxis and how different proteins in the FA are involved remain unclear. Zyxin is a component of the FA that has been implicated in connecting the actin cytoskeleton to the FA. We have found that knocking down zyxin impaired NIH3T3 fibroblast's ability to sense and respond to changes in extracellular matrix in terms of their FA sizes, cell traction stress magnitudes and F-actin organization. Cell migration speed of zyxin knockdown fibroblasts was also independent of the underlying substrate rigidity, unlike wild type fibroblasts which migrated fastest at an intermediate substrate rigidity of 14 kPa. Wild type fibroblasts exhibited durotaxis by migrating toward regions of increasing substrate rigidity on polyacrylamide gels with substrate rigidity gradient, while zyxin knockdown fibroblasts did not exhibit durotaxis. Therefore, we propose zyxin as an essential protein that is required for rigidity sensing and durotaxis through modulating FA sizes, cell traction stress and F-actin organization.

Dynamic swimming pattern of Pseudomonas aeruginosa near a vertical wall during initial attachment stages of biofilm formation.

Studying the swimming behaviour of bacteria in 3 dimensions (3D) allows us to understand critical biological processes, such as biofilm formation. It is still unclear how near wall swimming behaviour may regulate the initial attachment and biofilm formation. It is challenging to address this as visualizing the movement of bacteria with reasonable spatial and temporal resolution in a high-throughput manner is technically difficult. Here, we compared the near wall (vertical) swimming behaviour of P. aeruginosa (PAO1) and its mutants ΔdipA (reduced in swarming motility and increased in biofilm formation) and ΔfimX (deficient in twitching motility and reduced in biofilm formation) using our new imaging technique based on light sheet microscopy. We found that P. aeruginosa (PAO1) increases its speed and changes its swimming angle drastically when it gets closer to a wall. In contrast, ΔdipA mutant moves toward the wall with steady speed without changing of swimming angle. The near wall behavior of ΔdipA allows it to be more effective to interact with the wall or wall-attached cells, thus leading to more adhesion events and a larger biofilm volume during initial attachment when compared with PAO1. Furthermore, we found that ΔfimX has a similar near wall swimming behavior as PAO1. However, it has a higher dispersal frequency and smaller biofilm formation when compared with PAO1 which can be explained by its poor twitching motility. Together, we propose that near wall swimming behavior of P. aeruginosa plays an important role in the regulation of initial attachment and biofilm formation.

POPX2 phosphatase enhances topographical contact guidance for cell morphology and migration.

Topography mediated contact guidance affects multiple cell behaviors such as establishment of cellular morphology and migration. The direction of cell migration is associated with the establishment of cell polarity, which also affects the primary cilia in migrating cells. POPX2, a partner of PIX2, is involved in pathways essential to primary cilium formation, while over-expression of POPX2 has been reported to cause a loss of cell polarity during migration. This study aims to examine how topographical cues direct morphological changes, and how topography affects the process of cellular migration and primary cilium architecture, in the context of POPX2 over-expression. Thus, the effect of anisotropic topography, 2 µm grating pattern on tissue-culture polystyrene, was used as a contact guidance cue to investigate the migration and cell polarity of POPX2 overexpressing cells, in comparison to control NIH3T3 fibroblast cells. We report that POPX2 overexpressing NIH3T3 cells were more sensitive to surface topographical cues as the cells became more elongated. In addition, these cues also affected focal adhesion alignment of POPX2 overexpressing cells. Cell migration was further studied using wound closure assays, in which the 2 µm gratings were designed to be either perpendicular or parallel to wound-induced cell migration direction, which would be agonistic or antagonistic to cell migration, respectively. We observed that both POPX2 overexpressing cells' migration direction and migration rate were more significantly influenced by gratings direction compared to control NIH3T3 cells. The migration paths of POPX2 overexpressing cells become more direct in the presence of anisotropic topographical cues. Further, cilia and centrosome alignment, which is important in cell migration, was also affected by the direction of gratings during this migration process. Collectively, enhancement of NIH3T3 cell sensitivity towards surface topography through POPX2 overexpression might reflect one of the mechanisms that combine biochemical and mechanical cues for directional cell migration.

Partners in crime: POPX2 phosphatase and its interacting proteins in cancer.

Protein phosphorylation and dephosphorylation govern intracellular signal transduction and cellular functions. Kinases and phosphatases are involved in the regulation and development of many diseases such as Alzheimer's, diabetes, and cancer. While the functions and roles of many kinases, as well as their substrates, are well understood, phosphatases are comparatively less well studied. Recent studies have shown that rather than acting on fewer and more distinct substrates like the kinases, phosphatases can recognize specific phosphorylation sites on many different proteins, making the study of phosphatases and their substrates challenging. One approach to understand the biological functions of phosphatases is through understanding their protein-protein interaction network. POPX2 (Partner of PIX 2; also known as PPM1F or CaMKP) is a serine/threonine phosphatase that belongs to the PP2C family. It has been implicated in cancer cell motility and invasiveness. This review aims to summarize the different binding partners of POPX2 phosphatase and explore the various functions of POPX2 through its interactome in the cell. In particular, we focus on the impact of POPX2 on cancer progression. Acting via its different substrates and interacting proteins, POPX2's involvement in metastasis is multifaceted and varied according to the stages of metastasis.

Unraveling Heterogeneity in Transcriptome and Its Regulation Through Single-Cell Multi-Omics Technologies.

Cellular heterogeneity plays a pivotal role in tissue homeostasis and the disease development of multicellular organisms. To deconstruct the heterogeneity, a multitude of single-cell toolkits measuring various cellular contents, including genome, transcriptome, epigenome, and proteome, have been developed. More recently, multi-omics single-cell techniques enable the capture of molecular footprints with a higher resolution by simultaneously profiling various cellular contents within an individual cell. Integrative analysis of multi-omics datasets unravels the relationships between cellular modalities, builds sophisticated regulatory networks, and provides a holistic view of the cell state. In this review, we summarize the major developments in the single-cell field and review the current state-of-the-art single-cell multi-omic techniques and the bioinformatic tools for integrative analysis.

Parallel bimodal single-cell sequencing of transcriptome and chromatin accessibility.

Joint profiling of transcriptome and chromatin accessibility within single cells allows for the deconstruction of the complex relationship between transcriptional states and upstream regulatory programs determining different cell fates. Here, we developed an automated method with high sensitivity, assay for single-cell transcriptome and accessibility regions (ASTAR-seq), for simultaneous measurement of whole-cell transcriptome and chromatin accessibility within the same single cell. To show the utility of ASTAR-seq, we profiled 384 mESCs under naive and primed pluripotent states as well as a two-cell like state, 424 human cells of various lineage origins (BJ, K562, JK1, and Jurkat), and 480 primary cord blood cells undergoing erythroblast differentiation. With the joint profiles, we configured the transcriptional and chromatin accessibility landscapes of discrete cell states, uncovered linked sets of cis-regulatory elements and target genes unique to each state, and constructed interactome and transcription factor (TF)-centered upstream regulatory networks for various cell states.

A novel function of AAA-ATPase p97/VCP in the regulation of cell motility.

High level of the multifunctional AAA-ATPase p97/VCP is often correlated to the development of cancer; however, the underlying mechanism is not understood completely. Here, we report a novel function of p97/VCP in actin regulation and cell motility. We found that loss of p97/VCP promotes stabilization of F-actin, which cannot be reversed by actin-destabilizing agent, Cytochalasin D. Live-cell imaging demonstrated reduced actin dynamics in p97/VCP-knockdown cells, leading to compromised cell motility. We further examined the underlying mechanism and found elevated RhoA protein levels along with increased phosphorylation of its downstream effectors, ROCK, LIMK, and MLC upon the knockdown of p97/VCP. Since p97/VCP is indispensable in the ubiquitination-dependent protein degradation pathway, we investigated if the loss of p97/VCP hinders the protein degradation of RhoA. Knockdown of p97/VCP resulted in a higher amount of ubiquitinated RhoA, suggesting p97/VCP involvement in the proteasome-dependent protein degradation pathway. Finally, we found that p97/VCP interacts with FBXL19, a molecular chaperone known to guide ubiquitinated RhoA for proteasomal degradation. Reduction of p97/VCP may result in the accumulation of RhoA which, in turn, enhances cytoplasmic F-actin formation. In summary, our study uncovered a novel function of p97/VCP in actin regulation and cell motility via the Rho-ROCK dependent pathway which provides fundamental insights into how p97/VCP is involved in cancer development.

Phosphatase POPX2 interferes with cell cycle by interacting with Chk1.

Protein-protein interaction network analysis plays critical roles in predicting the functions of target proteins. In this study, we used a combination of SILAC-MS proteomics and bioinformatic approaches to identify Checkpoint Kinase 1 (Chk1) as a possible POPX2 phosphatase interacting protein. POPX2 is a PP2C phosphatase that has been implicated in cancer cell invasion and migration. From the Domain-Domain Interaction (DDI) database, we first determined that the PP2C phosphatase domain interacts with Pkinase domain. Subsequently, 46 proteins with Pkinase domain were identified from POPX2 SILAC-MS data. We then narrowed down the leads and confirmed the biological interaction between Chk1 and POPX2. We also found that Chk1 is a substrate of POPX2. Chk1 is a key regulator of the cell cycle and is activated when the cell suffers DNA damage. Our approach has led us to identify POPX2 as a regulator of Chk1 and can interfere with the normal function of Chk1 at G1-S transition of the cell cycle in response to DNA damage.

Correction: POPX2 is a novel LATS phosphatase that regulates the Hippo pathway.

[This corrects the article DOI: 10.18632/oncotarget.26689.].

POPX2 is a novel LATS phosphatase that regulates the Hippo pathway.

The Hippo pathway regulates cell proliferation, survival, apoptosis and differentiation. During carcinogenesis, members of the Hippo pathway are mutated to avoid anoikis and promote anchorage independent growth. Although many regulators of the Hippo pathway have been reported, negative regulators of the hippo kinases are not well studied. Through an interactome screen, we found that POPX2 phosphatase interacts with several of the Hippo pathway core kinases, including LATS1 which is the direct kinase regulating the transcription co-activators, YAP and TAZ. Phosphorylated YAP/TAZ are retained in the cytoplasm and prevented from translocation into the nucleus to activate transcription of target genes. We found that POPX2 could dephosphorylate LATS1 on Threonine-1079, leading to inactivation of LATS1 kinase. As a result, YAP/TAZ are not phosphorylated and are able to translocate into the nucleus to activate target genes involved in cell proliferation. Furthermore, POPX2 knock-out using CRISPR in the highly metastatic MDA-MB-231 breast cancer cells results in decreased cell proliferation and impairment of anchorage independent growth. We propose that POPX2 act as a suppressor of the Hippo pathway through LATS1 dephosphorylation and inactivation.

LIM kinase1 regulates mitotic centrosome integrity via its activity on dynein light intermediate chains.

Abnormal centrosome number and function have been implicated in tumour development. LIM kinase1 (LIMK1), a regulator of actin cytoskeleton dynamics, is found to localize at the mitotic centrosome. However, its role at the centrosome is not fully explored. Here, we report that LIMK1 depletion resulted in multi-polar spindles and defocusing of centrosomes, implicating its involvement in the regulation of mitotic centrosome integrity. LIMK1 could influence centrosome integrity by modulating centrosomal protein localization at the spindle pole. Interestingly, dynein light intermediate chains (LICs) are able to rescue the defects observed in LIMK1-depleted cells. We found that LICs are potential novel interacting partners and substrates of LIMK1 and that LIMK1 phosphorylation regulates cytoplasmic dynein function in centrosomal protein transport, which in turn impacts mitotic spindle pole integrity.

Enhanced Delta-Notch Lateral Inhibition Model Incorporating Intracellular Notch Heterogeneity and Tension-Dependent Rate of Delta-Notch Binding that Reproduces Sprouting Angiogenesis Patterns.

Endothelial cells adopt unique cell fates during sprouting angiogenesis, differentiating into tip or stalk cells. The fate selection process is directed by Delta-Notch lateral inhibition pathway. Classical Delta-Notch models produce a spatial pattern of tip cells separated by a single stalk cell, or the salt-and-pepper pattern. However, classical models cannot explain alternative tip-stalk patterning, such as tip cells that are separated by two or more stalk cells. We show that lateral inhibition models involving only Delta and Notch proteins can also recapitulate experimental tip-stalk patterns by invoking two mechanisms, specifically, intracellular Notch heterogeneity and tension-dependent rate of Delta-Notch binding. We introduce our computational model and analysis where we establish that our enhanced Delta-Notch lateral inhibition model can recapitulate a greater variety of tip-stalk patterning which is previously not possible using classical lateral inhibition models. In our enhanced Delta-Notch lateral inhibition model, we observe the existence of a hybrid cell type displaying intermediate tip and stalk cells' characteristics. We validate the existence of such hybrid cells by immuno-staining of endothelial cells with tip cell markers, Delta and CD34, which substantiates our enhanced model.

POPX2 phosphatase regulates apoptosis through the TAK1-IKK-NF-κB pathway.

Chemoresistance is one of the leading causes that contributes to tumor relapse and poor patient outcome after several rounds of drug therapy. The causes of chemoresistance are multi-factorial. Ultimately, it is the balance of pro- and anti-apoptotic activities in the cells. We have previously reported links between POPX2 serine/threonine phosphatase with cell motility and invasiveness of breast cancer cells. Here, we show that POPX2 plays a role in the regulation of apoptosis. The effect of POPX2 on apoptosis centers on the inactivation of TGF-ß activated kinase (TAK1). TAK1 is essential for several important biological functions including innate immunity, development and cell survival. We find that POPX2 interacts directly with TAK1 and is able to dephosphorylate TAK1. Cells with lower levels of POPX2 exhibit higher TAK1 activity in response to etoposide (VP-16) treatment. This subsequently leads to increased translocation of NF-κB from the cytosol to the nucleus. Consequently, NF-κB-mediated transcription of anti-apoptotic proteins is upregulated to promote cell survival. On the other hand, cells with higher levels of POPX2 are more vulnerable to apoptosis induced by etoposide. Our data demonstrate that POPX2 is a negative regulator of TAK1 signaling pathway and modulates apoptosis through the regulation of TAK1 activity. As inhibition of TAK1 has been proposed to reduce chemoresistance and increase sensitivity to chemotherapy in certain types of cancer, modulation of POPX2 levels may provide an additional avenue and consideration in fine-tuning therapeutic response.

Phosphatase POPX2 Exhibits Dual Regulatory Functions in Cancer Metastasis.

Cancer metastasis is a complex mechanism involving multiple processes. Previously, our integrative proteome, transcriptome, and phosphoproteome study reported that the levels of serine/threonine phosphatase POPX2 were positively correlated with cancer cell motility through modulating MAPK signaling. Surprisingly, here we found that POPX2 knockdown cells induced more numerous and larger tumor nodules in lungs in longer term animal studies. Interestingly, our analysis of DNA microarray data from cancer patient samples that are available in public databases shows that low POPX2 expression is linked to distant metastasis and poor survival rate. These observations suggest that lower levels of POPX2 may favor tumor progression in later stages of metastasis. We hypothesize that POPX2 may do so by modulation of angiogenesis. Secretome analysis of POPX2-knockdown MDA-MB-231 cells using LC-MS/MS-based SILAC quantitative proteomics and cytokine array show that silencing of POPX2 leads to increased secretion of exosomes, which may, in turn, induce multiple pro-angiogenic cytokines. This study, combined with our previous findings, suggests that a single ubiquitously expressed phosphatase POPX2 influences cancer metastasis via modulating multiple biological processes including MAPK signaling and exosome cytokine secretion.

Aurora kinase-induced phosphorylation excludes transcription factor RUNX from the chromatin to facilitate proper mitotic progression.

The Runt-related transcription factors (RUNX) are master regulators of development and major players in tumorigenesis. Interestingly, unlike most transcription factors, RUNX proteins are detected on the mitotic chromatin and apparatus, suggesting that they are functionally active in mitosis. Here, we identify key sites of RUNX phosphorylation in mitosis. We show that the phosphorylation of threonine 173 (T173) residue within the Runt domain of RUNX3 disrupts RUNX DNA binding activity during mitotic entry to facilitate the recruitment of RUNX proteins to mitotic structures. Moreover, knockdown of RUNX3 delays mitotic entry. RUNX3 phosphorylation is therefore a regulatory mechanism for mitotic entry. Cancer-associated mutations of RUNX3 T173 and its equivalent in RUNX1 further corroborate the role of RUNX phosphorylation in regulating proper mitotic progression and genomic integrity.

The Regulation of Cellular Responses to Mechanical Cues by Rho GTPases.

The Rho GTPases regulate many cellular signaling cascades that modulate cell motility, migration, morphology and cell division. A large body of work has now delineated the biochemical cues and pathways, which stimulate the GTPases and their downstream effectors. However, cells also respond exquisitely to biophysical and mechanical cues such as stiffness and topography of the extracellular matrix that profoundly influence cell migration, proliferation and differentiation. As these cellular responses are mediated by the actin cytoskeleton, an involvement of Rho GTPases in the transduction of such cues is not unexpected. In this review, we discuss an emerging role of Rho GTPase proteins in the regulation of the responses elicited by biophysical and mechanical stimuli.