Collective cell migration (CCM) drives physiological processes such as embryogenesis, vascular sprouting, and wound healing, but is also a major contributor to cancer metastasis. Ras homolog family member A (RhoA) drives CCM by modulating actomyosin cytoskeletal activity. RhoA activity is tightly spatiotemporally controlled during CCM, but whether these activation dynamics are shared by its downstream effectors is unknown. To investigate the role of RhoA's downstream effector Rho-associated kinase (ROCK) in collectively migrating fibroblasts, we developed a new single color FRET-based ROCK sensor, the Rho kinase activity reporter (RhoKAR) sensor.

Ras homolog family member A (RhoA) acts as a signaling hub in many cellular processes, including cytoskeletal dynamics, division, migration, and adhesion. RhoA activity is tightly spatiotemporally controlled, but whether downstream effectors share these activation dynamics is unknown. We developed a novel single-color FRET biosensor to measure Rho-associated kinase (ROCK) activity with high spatiotemporal resolution in live cells. We report the validation of the Rho-Kinase Activity Reporter (RhoKAR) biosensor. RhoKAR activation was specific to ROCK activity and was insensitive to PKA activity. We then assessed the mechanisms of ROCK activation in mouse fibroblasts. Increasing intracellular calcium with ionomycin increased RhoKAR activity and depleting intracellular calcium with EGTA decreased RhoKAR activity. We also investigated the signaling intermediates in this process. Blocking calmodulin or CaMKII prevented calcium-dependent activation of ROCK. These results indicate that ROCK activity is increased by calcium in fibroblasts and that this activation occurs downstream of CaM/CaMKII.

Collective cell migration (CCM) plays an important role in embryogenesis, vascular sprouting, and wound healing, but is also a significant driver of cancer metastasis. Ras homolog family member A (RhoA) facilitates CCM by modulating contraction of the actomyosin cytoskeleton. Given the precise spatiotemporal regulation of RhoA during CCM, we wanted to investigate the activity of downstream effector Rho-associated kinase (ROCK) in collectively migrating fibroblasts. Using a new FRET-based ROCK biosensor, we have observed calcium-dependent activation of ROCK. We have previously demonstrated blunting of CCM in response to pharmacological inhibition of ROCK or depletion of intracellular calcium with 100 µM EGTA. To study the role of gap junction signaling in calcium-dependent activation of ROCK during CCM, carbenoxolone, a gap junction blocker, was applied to collectively migrating cells following scratch wounding. Carbenoxolone treatment blunted CCM, as well as decreased ROCK activity and intracellular calcium levels at corresponding time points. Treatment with PQ7, a gap junction activator, caused modest increases in CCM. These results indicate that calcium signaling through gap junctions drives ROCK activity in the context of collective cell migration.

Collective cell migration (CCM) is a significant driver of cancer metastasis and invasion. Ras homolog family member A (RhoA) facilitates CCM by modulating contraction of the actomyosin cytoskeleton. Given the tight control over RhoA activity in space and time, it is unknown whether the downstream targets share similar activation patterns. To measure Rho-associated kinase (ROCK) activity in migrating cells, we developed a new FRET-based biosensor. Here we report the validation of a FLuorescence Anisotropy Reporter (FLARE)-type ROCK sensor. Activation of the ROCK-FLARE sensor was also specific to ROCK activity and insensitive to activation of the PKA pathway. We then assessed the mechanism of ROCK activation in collectively migrating fibroblasts. Increased cytoplasmic activated the ROCK sensor. Further, depletion of calcium levels with 100 µM EGTA decreased ROCK activity and impaired CCM. Inhibition of ROCK with Y27362 also blunted CCM behavior in fibroblasts. Thus, ROCK activity in collectively migrating fibroblasts is downstream of calcium release, likely from intracellular stores.

We have shown that SERCA1 activity is inhibited by small ankyrin 1 (sAnk1), a ∼20kDa transmembrane protein that stabilizes the network SR in skeletal muscle and that can form a 3-way complex with SERCA1 and sarcolipin (SLN). Here, we show that sAnk1 also associates with phospholamban (PLN) and can form a 3-way complex with PLN and SERCA1. Association of PLN and sAnk1 was demonstrated by bimolecular fluorescent complementation (BiFC), sAnk1 with the C-terminal of half-Venus and PLN with the N-terminal of half-Venus. Independently, neither show fluorescence, but when co-expressed these two constructs were fluorescent, indicating reconstitution of Venus. A control half Venus dysferlin construct did not fluorescence when expressed with the sAnk1 construct. The BiFC was used with Cerulean-SERCA1 to study three-way interaction. These studies confirm that sAnk1 associates specifically with PLN, and the two proteins together complex with SERCA1. We are pursuing co-immunoprecipitation studies and ATPase assays to further study the effects of sAnk1 on PLN-SERCA1 complexes. Our results show sAnk1 and PLN can work together to lower SERCA1 activity. These results have significant implications for the development of therapeutic approaches to treat a variety of diseases linked to calcium misregulation such as muscular dystrophies and cardiopathies.

Förster resonance energy transfer (FRET) between fluorophores of the same species was recognized in the early to mid-1900s, well before modern heterotransfer applications. Recently, homotransfer FRET principles have re-emerged in biosensors that incorporate genetically encoded fluorescent proteins. Homotransfer offers distinct advantages over the standard heterotransfer FRET method, some of which are related to the use of fluorescence polarization microscopy to quantify FRET between two fluorophores of identical color. These include enhanced signal-to-noise, greater compatibility with other optical sensors and modulators, and new design strategies based upon the clustering or dimerization of singly-labeled sensors. Here, we discuss the theoretical basis for measuring homotransfer using polarization microscopy, procedures for data collection and processing, and we review the existing genetically-encoded homotransfer biosensors.

The release of insulin from the pancreas is tightly controlled by glucokinase (GCK) activity that couples β-cell metabolism to changes in blood sugar. Despite having only a single glucose-binding site, GCK displays positive glucose cooperativity. Ex vivo structural studies have identified several potential protein conformations with varying levels of enzymatic activity, yet it is unclear how living cells regulate GCK cooperativity. To better understand the cellular regulation of GCK activation, we developed a homotransfer Förster resonance energy transfer (FRET) GCK biosensor and used polarization microscopy to eliminate fluorescence crosstalk from FRET quantification and improve the signal-to-noise ratio. This approach enhanced sensor contrast compared to that seen with the heterotransfer FRET GCK reporter and allowed observation of individual GCK states using an automated method to analyze FRET data at the pixel level. Mutations known to activate and inhibit GCK activity produced distinct anisotropy distributions, suggesting that at least two conformational states exist in living cells. A high glucose level activated the biosensor in a manner consistent with GCK's enzymology. Interestingly, glucose-free conditions did not affect GCK biosensor FRET, indicating that there is a single low-activity state, which is counter to proposed structural models of GCK cooperativity. Under low-glucose conditions, application of chemical NO donors efficiently shifted GCK to the more active conformation. Notably, GCK activation by mutation, a high glucose level, a pharmacological GCK activator, or S-nitrosylation all shared the same FRET distribution. These data suggest a simplified model for GCK activation in living cells, where post-translational modification of GCK by S-nitrosylation facilitates a single conformational transition that enhances GCK enzymatic activity.

Although cytokine-dependent dynamics of nuclear factor κB (NF-κB) are known to encode information that regulates cell fate decisions, it is unclear whether single-cell responses are switch-like or encode more information about cytokine dose. Here, we measure the dynamic subcellular localization of NF-κB in response to a range of tumor necrosis factor (TNF) stimulation conditions to determine the prevailing mechanism of single-cell dose discrimination. Using an information theory formalism that accounts for signaling dynamics and non-responsive cell subpopulations, we find that the information transmission capacity of single cells exceeds that predicted from a switch-like response. Instead, we observe that NF-κB dynamics within single cells contain sufficient information to encode multiple, TNF-dependent cellular states, and have an activation threshold that varies across the population. By comparing single-cell responses to an internal, experimentally observed reference, we demonstrate that cells can grade responses to TNF across several orders of magnitude in concentration. This suggests that cells contain additional control points to fine-tune their cytokine responses beyond the decision to activate.

SERCA1, the sarco(endo)plasmic reticulum Ca2+-ATPase of skeletal muscle, is essential for muscle relaxation and maintenance of low resting Ca2+ levels in the myoplasm. We recently reported that small ankyrin 1 (sAnk1) interacts with the sarco(endo)plasmic reticulum Ca2+-ATPase in skeletal muscle (SERCA1) to inhibit its activity. We also showed that this interaction is mediated at least in part through sAnk1's transmembrane domain in a manner similar to that of sarcolipin (SLN). Earlier studies have shown that SLN and phospholamban, the other well studied small SERCA-regulatory proteins, oligomerize either alone or together. As sAnk1 is coexpressed with SLN in muscle, we sought to determine whether these two proteins interact with one another when coexpressed exogenously in COS7 cells. Coimmunoprecipitation (coIP) and anisotropy-based FRET (AFRET) assays confirmed this interaction. Our results indicated that sAnk1 and SLN can associate in the sarcoplasmic reticulum membrane and after exogenous expression in COS7 cells in vitro but that their association did not require endogenous SERCA2. Significantly, SLN promoted the interaction between sAnk1 and SERCA1 when the three proteins were coexpressed, and both coIP and AFRET experiments suggested the formation of a complex consisting of all three proteins. Ca2+-ATPase assays showed that sAnk1 ablated SLN's inhibition of SERCA1 activity. These results suggest that sAnk1 interacts with SLN both directly and in complex with SERCA1 and reduces SLN's inhibitory effect on SERCA1 activity.

ABSTRACTBoth CD4+ T lymphocytes and macrophages are the major targets of human immunodeficiency virus type 1 (HIV‐1); however, they respond differently to HIV‐1 infection. We hypothesized that HIV‐1 infection alters gene expression in CD4+ T cells and monocyte‐derived macrophages (MDMs) in a cell specific manner and microRNAs (miRNAs) in part play a role in cell‐specific gene expression. Results indicate that 183 and 31 genes were differentially regulated in HIV‐1 infected CD4+ T cells and MDMs, respectively, compared to their mock‐infected counterparts. Among the differentially expressed genes, cell cycle regulatory gene, p21 (CDKN1A) was upregulated in virus infected CD4+ T cells both at the mRNA and protein level in CD4+ T cells, whereas no consistent change was observed in MDMs. Productively infected CD4+ T cells express higher amount of p21 compared to bystander cells. In determining the mechanism(s) of cell type specific regulation of p21, we found that the miRNAs miR‐106b and miR‐20a that target p21 were specifically downregulated in HIV‐1 infected CD4+ T cells. Overexpression of these two miRNAs reduced p21 expression significantly in HIV‐1 infected CD4+ T cells. These findings provide a potential mechanism, by which, HIV‐1 could exploit host cellular machineries to regulate selective gene expression in target cells. J. Cell. Biochem. 117: 1902–1912, 2016. © 2016 Wiley Periodicals, Inc.

Background Latent HIV-1 reservoirs are identified as one of the major challenges to achieve HIV-1 cure. Currently available strategies are associated with wide variability in outcomes both in patients and CD4 + T cell models. This underlines the critical need to develop innovative strategies to predict and recognize ways that could result in better reactivation and eventual elimination of latent HIV-1 reservoirs. Results and discussion In this study, we combined genome wide transcriptome datasets post activation with Systems Biology approach (Signaling and Dynamic Regulatory Events Miner, SDREM analyses) to reconstruct a dynamic signaling and regulatory network involved in reactivation mediated by specific activators using a latent cell line. This approach identified several critical regulators for each treatment, which were confirmed in follow-up validation studies using small molecule inhibitors. Results indicate that signaling pathways involving JNK and related factors as predicted by SDREM are essential for virus reactivation by suberoylanilide hydroxamic acid. ERK1/2 and NF-κB pathways have the foremost role in reactivation with prostratin and TNF-α, respectively. JAK-STAT pathway has a central role in HIV-1 transcription. Additional evaluation, using other latent J-Lat cell clones and primary T cell model, also confirmed that many of the cellular factors associated with latency reversing agents are similar, though minor differences are identified. JAK-STAT and NF-κB related pathways are critical for reversal of HIV-1 latency in primary resting T cells. Conclusion These results validate our combinatorial approach to predict the regulatory cellular factors and pathways responsible for HIV-1 reactivation in latent HIV-1 harboring cell line models. JAK-STAT have a role in reversal of latency in all the HIV-1 latency models tested, including primary CD4 + T cells, with additional cellular pathways such as NF-κB, JNK and ERK 1/2 that may have complementary role in reversal of HIV-1 latency.