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.

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.

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.

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.

POPX2 phosphatase regulates the KIF3 kinesin motor complex.

The kinesin motors are important in the regulation of cellular functions such as protein trafficking, spindle organization and centrosome separation. In this study, we have identified POPX2, a serine-threonine phosphatase, as an interacting partner of the KAP3 subunit of the kinesin-2 motor. The kinesin-2 motor is a heterotrimeric complex composed of KIF3A, KIF3B motor subunits and KAP3, the non-motor subunit, which binds the cargo. Here we report that the phosphatase POPX2 is a negative regulator of the trafficking of N-cadherin and other cargoes; consequently, it markedly influences cell-cell adhesion. POPX2 affects trafficking by determining the phosphorylation status of KIF3A at serine 690. This is consistent with the observation that the KIF3A-S690A mutant is defective in cargo trafficking. Our studies also implicate CaMKII as the kinase that phosphorylates KIF3A at serine 690. These results strongly suggest that POPX2 and CaMKII are a phosphatase-kinase pair that regulates kinesin-mediated transport and cell-cell adhesion.

TPPP acts downstream of RhoA-ROCK-LIMK2 to regulate astral microtubule organization and spindle orientation.

The actin cytoskeleton in eukaryotic cells undergoes drastic rearrangement during mitosis. The changes to the actin cytoskeleton are most obvious in the adherent cells, where the actin stress fibres are disassembled, and the cortical actin network becomes more prominent with concomitant increase in cell rigidity as cells round up and enter mitosis. Although the regulatory connections between the actin cytoskeleton and the early mitotic events are apparent, the mechanisms that govern these links are not well understood. Here, we report that LIMK1 and LIMK2, the downstream effectors of RhoA and ROCK, regulate centrosome integrity and astral microtubule organization, respectively. Surprisingly, LIMK1 and cofilin are not involved downstream of RhoA and ROCK in the regulation of astral microtubule organization. Instead, we find that LIMK2 acts through TPPP in the regulation of astral microtubule organization, whereas both LIMK1 and LIMK2 affect centrosome focusing. Both phenotypes are tightly coupled to spindle orientation in the mitotic cells. Thus, our results reveal a new regulatory link between the actin cytoskeleton and the mitotic spindle during the early stages of mitosis.

Integrative transcriptome and proteome study to identify the signaling network regulated by POPX2 phosphatase.

POPX2 is a serine/threonine phosphatase belonging to the protein phosphatase 2C (PP2C) family that has been found to be elevated in invasive breast cancer cells. Silencing of POPX2 results in lower cell motility and invasiveness. The molecular mechanism of POPX2-regulated cell motility is not well understood. To identify the relevant signaling pathways, we investigated the transcriptome and proteome of POPX2-knockdown MDA-MB-231 breast cancer cells. Our data suggest that POPX2 might be involved in the regulation of focal adhesions and cytoskeleton dynamics through the regulation of MAP kinase (MAPK1/3) and glycogen synthase kinase 3 (GSK3α/ß) activities. Silencing POPX2 alters phosphorylation levels of MAPK1/3 and GSK3α/ß and results in reduced activity of these kinases. Both MAPK and GSK3 are known to regulate the activities of transcription factors. MAPK1/3 are also implicated in the phosphorylation of stathmin. The level of phospho-stathmin was found to be lower in POPX2 knockdown cells. As phosphorylation of stathmin inhibits its microtubule severing activity, we observed less stable microtubules in POPX2 knockdown cells. Taken together, our data suggest that POPX2 might regulate cell motility through its regulation of the MAPK1/3, leading to changes in the cytoskeleton and cell motility.

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.

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.

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.

Investigating circular dorsal ruffles through varying substrate stiffness and mathematical modeling.

Circular dorsal ruffles (CDRs) are transient actin-rich ringlike structures that form on the dorsal surface of growth-factor stimulated cells. However, the dynamics and mechanism of formation of CDRs are still unknown. It has been observed that CDR formation leads to stress fibers disappearing near the CDRs. Because stress fiber formation can be modified by substrate stiffness, we examined the effect of substrate stiffness on CDR formation by seeding NIH 3T3 fibroblasts on glass and polydimethylsiloxane substrates of varying stiffnesses from 20 kPa to 1800 kPa. We found that increasing substrate stiffness increased the lifetime of the CDRs. We developed a mathematical model of the signaling pathways involved in CDR formation to provide insight into this lifetime and size dependence that is linked to substrate stiffness via Rac-Rho antagonism. From the model, increasing stiffness raised mDia1-nucleated stress fiber formation due to Rho activation. The increased stress fibers present increased replenishment of the G-actin pool, therefore prolonging Arp2/3-nucleated CDR formation due to Rac activation. Negative feedback by WAVE-related RacGAP on Rac explained how CDR actin propagates as an excitable wave, much like wave propagation in other excitable medium, e.g., nerve signal transmission.

Investigation of POPX2 phosphatase functions by comparative phosphoproteomic analysis.

Identifying the substrates and biochemical pathway regulated by phosphatases has always been more challenging than finding those regulated by kinases. Here, we report the use of phosphoproteomic methods to analyse the pathways regulated by POPX2 (partner of PIX 2) phosphatase. POPX2 is a serine/threonine phosphatase, found in many cancer types. The levels of the POPX2 have been found to be up-regulated in the more invasive breast cancer cells compared with non-invasive ones. Our observations also suggest that POPX2 level is positively correlated with cell motility. Thus, finding substrates or pathways regulated by POPX2 will help to elucidate the regulatory mechanism of cancer cell motility and invasiveness. We have also developed and validated a protocol using electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) to enrich the phosphopeptides followed by LC-MS/MS to allow comparison between the phosphoproteomes of control and POPX2 overexpressing cells. With this approach, we were able to identify a biochemical pathway through which POPX2 exerts its apparent cellular function: the regulation of activity of glycogen synthase kinase-3, which in turn modulates extracellular signal-regulated kinase and cell motility.

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.

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.

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.

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.

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.

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.

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.

Rac1 GTPase is activated by hepatitis B virus replication--involvement of HBX.

Hepatitis B virus (HBV) is a causative agent for liver diseases including hepatocellular carcinoma. Understanding its interactions with cellular proteins is critical in the elucidation of the mechanisms of disease progression. Using a cell-based HBV replication system, we showed that HBV replication in HepG2 cells resulted in a cellular morphological changes displaying membrane rufflings and lamellipodia like structures reminiscent of cells expressing constitutively activated Rac1. We also showed that activated Rac1 resulted in increased viral replication. HBV replication specifically activated wild type Rac1, but not Cdc42. The Rac1 activation by HBV replication also resulted in the phosphorylation of ERK1/2 and AKT, the downstream targets of Rac1 signaling cascade. The smallest HBV viral protein, HBX, was able to activate the endogenous Rac1 and induce membrane ruffling when transfected into cells. Significantly, HBX was found to directly interact with a Rac1 nucleotide exchange factor (betaPIX) through a SH3 binding motif. Taken together, we have shown the interaction of HBV with the Rho GTPase, affecting cell morphology through the Rac1 activation pathway. HBV may possibly make use of an activated Rac1 signaling pathway for increased replication and resultant metastatic effects.