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This study explores the frictional dynamics in wire-on-V-groove contacts, particularly in slip rings, by examining the effects of contact geometry and loading stiffness. Our experimental setup, using three different custom tribometers, allowed precise control and measurement of these variables. Results show a significant deviation of the friction coefficient from traditional Amonton–Coulomb predictions, highlighting an increased sensitivity to the aforementioned factors. These findings challenge the conventional understanding of tribology, emphasizing the need for a refined theoretical model that better accounts for the complexities in V-groove contacts. The study's implications are far-reaching, particularly in improving the design and reliability of slip rings in high-precision instruments and satellite technologies.

The friction peak that occurs in tire-road sliding when the contact changes from wet to dry was previously attributed to capillary cohesion, van der Waals attraction, and surface roughness, but the detailed mechanisms have yet to be revealed. In this study, friction and static contact experiments were conducted using a custom-built in situoptical microtribometer, which allowed us to investigate the evolution of the friction, normal load, and contact area between a polydimethylsiloxane (PDMS) film and a silicon nitride ball during water volatilization. The friction coefficient increased by 100%, and the normal force dropped by 30% relative to those in the dry condition during the wet-to-dry transition. In static contact experiments, the probe indentation depth increased, and the normal load decreased by ∼60% as the water evaporated. Combining the friction and static contact results, we propose that the large friction peak that appeared in this study can be attributed to the combined effects of increased adhesive capillary force and increased plowing during the wet-to-dry transition.

Cancer cells are known for their ability to adapt variable metabolic programs depending on the availability of specific nutrients. Our previous studies have shown that uptake of fatty acids alters cellular metabolic pathways in colon cancer cells to favor fatty acid oxidation. Here, we show that fatty acids activate Drp1 to promote metabolic plasticity in cancer cells. Uptake of fatty acids (FAs) induces mitochondrial fragmentation by promoting ERK-dependent phosphorylation of Drp1 at the S616 site. This increased phosphorylation of Drp1 enhances its dimerization and interaction with Mitochondrial Fission Factor (MFF) at the mitochondria. Consequently, knockdown of Drp1 or MFF attenuates fatty acid-induced mitochondrial fission. In addition, uptake of fatty acids triggers mitophagy via a Drp1- and p62-dependent mechanism to protect mitochondrial integrity. Moreover, results from metabolic profiling analysis reveal that silencing Drp1 disrupts cellular metabolism and blocks fatty acid-induced metabolic reprograming by inhibiting fatty acid utilization. Functionally, knockdown of Drp1 decreases Wnt/β-catenin signaling by preventing fatty acid oxidation-dependent acetylation of β-catenin. As a result, Drp1 depletion inhibits the formation of tumor organoids in vitro and xenograft tumor growth in vivo. Taken together, our study identifies Drp1 as a key mediator that connects mitochondrial dynamics with fatty acid metabolism and cancer cell signaling.

Capillary forces of a shearing liquid bridge can significantly affect the friction and adhesion of interacting surfaces, but the underlying mechanisms remain unclear. We custom built a surface force apparatus (SFA, ±2 μN) equipped with in situoptical microscopy and performed normal and lateral force measurements on a reciprocating water bridge formed between two flat plates. A modified wedge method was developed to correct the unique force measurement errors caused by the changing bridge geometry and position. The results found (1) strong linear relations among the bridge shear displacement, the cosine difference between the left and right contact angles, and the lateral adhesion force and (2) the normal adhesion force increased monotonically up to 13% as the bridge geometry approached its axisymmetric state. Quasi-static force analyses based on a newly developed decahedral model showed good agreement with the experiments and improved accuracy compared with that of cylindrical or rectangular column models previously proposed in the literature. Although limited in certain aspects, this study may (1) prove helpful to the design and analysis of liquid bridge force experiments on platforms similar to the SFA used in this study and (2) help to bridge the gap between friction and liquid bridge physics in the literature.

Wnt signaling dysregulation promotes tumorigenesis in colorectal cancer (CRC). We investigated the role of PTPRF, a receptor-type tyrosine phosphatase, in regulating Wnt signaling in CRC. Knockdown of PTPRF decreased cell proliferation in patient-derived primary colon cancer cells and established CRC cell lines. In addition, the rate of proliferation as well as colony formation ability were significantly decreased in tumor organoids grown in 3D, whereas the number of differentiated tumor organoids were markedly increased. Consistently, knockdown of PTPRF resulted in a decrease in the expression of genes associated with cancer stem cells downstream of Wnt/β-catenin signaling. Treating PTPRF knockdown cells with GSK3 inhibitor rescued the expression of Wnt target genes suggesting that PTPRF functions upstream of the β-catenin destruction complex. PTPRF was found to interact with LRP6 and silencing PTPRF largely decreased the activation of LRP6. Interestingly, this PTPRF-mediated activation of Wnt signaling was blocked in cells treated with clathrin endocytosis inhibitor. Furthermore, knockdown of PTPRF inhibited xenograft tumor growth in vivo and decreased the expression of Wnt target genes. Taken together, our studies identify a novel role of PTPRF as an oncogenic protein phosphatase in supporting the activation of Wnt signaling in CRC.

Cancer cells are known to upregulate aerobic glycolysis to promote growth, proliferation, and survival. However, the role of mitochondrial respiration in tumorigenesis remains elusive. Here we report that inhibition of mitochondrial function by silencing TFAM, a key transcription factor essential for mitochondrial DNA (mtDNA) replication and the transcription of mtDNA-encoded genes, markedly reduced tumor-initiating potential of colon cancer cells. Knockdown of TFAM significantly decreased mitochondrial respiration in colon cancer cells; however, the cellular levels of ATP remained largely unchanged as a result of increased glycolysis. This metabolic alteration rendered cancer cells highly susceptible to glucose deprivation. Interestingly, upregulation of glycolysis was independent of hypoxia-inducible factor-1 (HIF1) as TFAM knockdown cells fail to stabilize HIF1α under hypoxic conditions. Moreover, knockdown of TFAM results in decreased expression of genes-associated cancer stem cells downstream of Wnt/β-catenin signaling. Metabolic analysis reveals that the level of α-ketoglutarate (α-KG) was significantly upregulated in TFAM knockout cells. Silencing of prolyl hydroxylase domain-containing protein 2 (PHD2), a α-KG-dependent dioxyenase, rescued the expression of target genes of both HIF1α and Wnt/β-catenin. Furthermore, intestinal-specific knockout of TFAM prevents tumor formation in Apc-mutant mouse models of colon cancer. Taken together, our findings identify a novel role of mitochondria-mediated retrograde signaling in regulating Wnt signaling and tumor initiation in colon cancer.

Autophagy is a highly regulated evolutionarily conserved metabolic process induced by stress and energy deprivation. Here, we show that DNA polymerase gamma (Polγ) deficiency activates a selective prosurvival autophagic response via mitochondria-mediated reactive oxygen species (ROS) signaling and the mammalian target of rapamycin complex 2 (mTORC2) activities. In keratinocytes, Polγ deficiency causes metabolic adaptation that triggers cytosolic sensing of energy demand for survival. Knockdown of Polγ causes mitochondrial stress, decreases mitochondrial energy production, increases glycolysis, increases the expression of autophagy-associated genes, and enhances AKT phosphorylation and cell proliferation. Deficiency of Polγ preferentially activates mTORC2 formation to increase autophagy and cell proliferation, and knocking down Rictor abrogates these responses. Overexpression of Rictor, but not Raptor, reactivates autophagy in Polγ-deficient cells. Importantly, inhibition of ROS by a mitochondria-selective ROS scavenger abolishes autophagy and cell proliferation. These results identify Rictor as a critical link between mitochondrial stress, ROS, and autophagy. They represent a major shift in our understanding of the prosurvival role of the mTOR complexes and highlight mitochondria-mediated ROS as a prosurvival autophagy regulator during cancer development.

Neurotensin (NT), a 13 amino-acid peptide, is predominantly released from enteroendocrine cells of the small bowel in response to fat ingestion. Free fatty acid receptors (FFARs) FFAR1 and FFAR4 regulate secretion of gut hormones and insulin. Here, we show that docosahexaenoic acid, a long-chain fatty acid, has the most dramatic effect on NT release. FFAR1 agonists slightly stimulate and FFAR4 agonists dramatically stimulate and amplify NT secretion. Double knockdown of FFAR1 and FFAR4 decreases NT release, whereas overexpression of FFAR4, but not FFAR1, increases NT release. Administration of cpdA, an FFAR4 agonist, but not TAK-875, a selective FFAR1 agonist, increases plasma NT levels and further increases olive oil-stimulated plasma NT levels. Inhibition of MAPK kinase (MEK)/ERK1/2 decreased fatty acid-stimulated NT release but increased AMP-activated protein kinase (AMPK) phosphorylation. In contrast, inhibition of AMPK further increased NT secretion and ERK1/2 phosphorylation mediated by FFAR1 or FFAR4. Our results indicate that FFAR4 plays a more critical role than FFAR1 in mediation of fat-regulated NT release and in inhibitory crosstalk between MEK/ERK1/2 and AMPK in the control of NT release downstream of FFAR1 and FFAR4.

Obesity and its associated comorbidities (for example, diabetes mellitus and hepatic steatosis) contribute to approximately 2.5 million deaths annually and are among the most prevalent and challenging conditions confronting the medical profession. Neurotensin (NT; also known as NTS), a 13-amino-acid peptide predominantly localized in specialized enteroendocrine cells of the small intestine and released by fat ingestion, facilitates fatty acid translocation in rat intestine, and stimulates the growth of various cancers. The effects of NT are mediated through three known NT receptors (NTR1, 2 and 3; also known as NTSR1, 2, and NTSR3, respectively). Increased fasting plasma levels of pro-NT (a stable NT precursor fragment produced in equimolar amounts relative to NT) are associated with increased risk of diabetes, cardiovascular disease and mortality; however, a role for NT as a causative factor in these diseases is unknown. Here we show that NT-deficient mice demonstrate significantly reduced intestinal fat absorption and are protected from obesity, hepatic steatosis and insulin resistance associated with high fat consumption. We further demonstrate that NT attenuates the activation of AMP-activated protein kinase (AMPK) and stimulates fatty acid absorption in mice and in cultured intestinal cells, and that this occurs through a mechanism involving NTR1 and NTR3 (also known as sortilin). Consistent with the findings in mice, expression of NT in Drosophila midgut enteroendocrine cells results in increased lipid accumulation in the midgut, fat body, and oenocytes (specialized hepatocyte-like cells) and decreased AMPK activation. Remarkably, in humans, we show that both obese and insulin-resistant subjects have elevated plasma concentrations of pro-NT, and in longitudinal studies among non-obese subjects, high levels of pro-NT denote a doubling of the risk of developing obesity later in life. Our findings directly link NT with increased fat absorption and obesity and suggest that NT may provide a prognostic marker of future obesity and a potential target for prevention and treatment.

Heat shock proteins play central roles in ensuring the correct folding and maturation of cellular proteins. Here we show that the heat shock protein Hsp70 has a novel role in prolonging the lifetime of activated protein kinase C. We identified Hsp70 in a screen for binding partners for the carboxyl terminus of protein kinase C. Co-immunoprecipitation experiments revealed that Hsp70 specifically binds the unphosphorylated turn motif (Thr(641) in protein kinase C beta II), one of three priming sites phosphorylated during the maturation of protein kinase C family members. The interaction of Hsp70 with protein kinase C can be abolished in vivo by co-expression of fusion proteins encoding the carboxyl terminus of protein kinase C or the carboxyl terminus of Hsp70. Pulse-chase experiments reveal that Hsp70 does not regulate the maturation of protein kinase C: the rate of processing by phosphorylation is the same in the presence or absence of disrupting constructs. Rather, Hsp70 prolongs the lifetime of mature protein kinase C; disruption of the interaction promotes the accumulation of matured and then dephosphorylated protein kinase C in the detergent-insoluble fraction of cells. Furthermore, studies with K562 cells reveal that disruption of the interaction with Hsp70 slows the protein kinase C beta II-mediated recovery of cells from PMA-induced growth arrest. Last, we show that other members of the AGC superfamily (Akt/protein kinase B and protein kinase A) also bind Hsp70 via their unphosphorylated turn motifs. Our data are consistent with a model in which Hsp70 binds the dephosphorylated carboxyl terminus of mature protein kinase C, thus stabilizing the protein and allowing re-phosphorylation of the enzyme. Disruption of this interaction prevents re-phosphorylation and targets the enzyme for down-regulation.

L-type Ca2+channels in native tissues have been found to contain a pore-forming α1subunit that is often truncated at the C terminus. However, the C terminus contains many important domains that regulate channel function. To test the hypothesis that C-terminal fragments may associate with and regulate C-terminal-truncated α1C(CaV1.2) subunits, we performed electrophysiological and biochemical experiments. In tsA201 cells expressing either wild type or C-terminal-truncated α1Csubunits in combination with a β2asubunit, truncation of the α1Csubunit by as little as 147 amino acids led to a 10–15-fold increase in currents compared with those obtained from control, full-length α1Csubunits. Dialysis of cells expressing the truncated α1Csubunits with C-terminal fragments applied through the patch pipette reconstituted the inhibition of the channels seen with full-length α1Csubunits. In addition, C-terminal deletion mutants containing a tethered C terminus also exhibited the C-terminal-induced inhibition. Immunoprecipitation assays demonstrated the association of the C-terminal fragments with truncated α1Csubunits. In addition, glutathioneS-transferase pull-down assays demonstrated that the C-terminal inhibitory fragment could associate with at least two domains within the C terminus. The results support the hypothesis the C- terminal fragments of the α1Csubunit can associate with C-terminal-truncated α1Csubunits and inhibit the currents through L-type Ca2+channels.

The function of protein kinase C family members depends on two tightly coupled phosphorylation mechanisms: phosphorylation of the activation loop by the phosphoinositide-dependent kinase, PDK-1, followed by autophosphorylation at two positions in the COOH terminus, the turn motif, and the hydrophobic motif. Here we address the molecular mechanisms underlying the regulation of protein kinase C βII by PDK-1. Co-immunoprecipitation studies reveal that PDK-1 associates preferentially with its substrate, unphosphorylated protein kinase C, by a direct mechanism. The exposed COOH terminus of protein kinase C provides the primary interaction site for PDK-1, with co-expression of constructs of the carboxyl terminus effectively disrupting the interaction in vivo. Disruption of this interaction promotes the autophosphorylation of protein kinase C, suggesting that the binding of PDK-1 to the carboxyl terminus protects it from autophosphorylation. Studies with constructs of the COOH terminus reveal that the intrinsic affinity of PDK-1 for phosphorylated COOH terminus is over an order of magnitude greater than that for unphosphorylated COOH terminus, contrasting with the finding that PDK-1 does not bind phosphorylated protein kinase C effectively. However, effective binding of the phosphorylated species can be induced by the activated conformation of protein kinase C. This suggests that the carboxyl terminus becomes masked following autophosphorylation, a process that can be reversed by the conformational changes accompanying activation. Our data suggest a model in which PDK-1 provides two points of regulation of protein kinase C: 1) phosphorylation of the activation loop, which is regulated by the intrinsic activity of PDK-1, and 2) phosphorylation of the carboxyl terminus, which is regulated by the release of PDK-1 to allow autophosphorylation.

The class C L-type calcium (Ca2+) channels have been implicated in many important physiological processes. Here, we have identified a mouse vacuolar H+-ATPase (V-ATPase) G2 subunit protein that bound to the C-terminal domain of the pore-forming α1C subunit using a yeast two-hybrid screen. Protein–protein interaction between the V-ATPase G subunit and the α1C subunit was confirmed using in vitro GST pull-down assays and coimmunoprecipitation from intact cells. Moreover, treatment of cells expressing L-type Ca2+ channels with a specific inhibitor of the V-ATPase blocked proper targeting of the channels to the plasma membrane.

We have previously demonstrated that formation of a complex between L-type calcium (Ca2+) channel α1C(CaV1.2) and β subunits was necessary to target the channels to the plasma membrane when expressed in tsA201 cells. In the present study, we identified a region in the C terminus of the α1Csubunit that was required for membrane targeting. Using a series of C-terminal deletion mutants of the α1Csubunit, a domain consisting of amino acid residues 1623–1666 (“targeting domain”) in the C terminus of the α1Csubunit has been identified to be important for correct targeting of L-type Ca2+channel complexes to the plasma membrane. Although cells expressing the wild-type α1Cand β2asubunits exhibited punctate clusters of channel complexes along the plasma membrane with little intracellular staining, co-expression of deletion mutants of the α1Csubunit that lack the targeting domain with the β2asubunit resulted in an intracellular localization of the channels. In addition, three other regions in the C terminus of the α1Csubunit that were downstream of residues 1623–1666 were found to contribute to membrane targeting of the L-type channels. Deletion of these domains in the α1Csubunit resulted in a reduction of plasma membrane-localized channels, and a concomitant increase in channels localized intracellularly. Taken together, these results have demonstrated that a targeting domain in the C terminus of the α1Csubunit was required for proper plasma membrane localization of the L-type Ca2+channels.

Although most L-type calcium channel α1Csubunits isolated from heart or brain are ∼190-kDa proteins that lack ∼50 kDa of the C terminus, the C-terminal domain is present in intact cells. To test the hypothesis that the C terminus is processed but remains functionally associated with the channels, expressed, full-length α1Csubunits were cleaved in vitroby chymotrypsin to generate a 190-kDa C-terminal truncated protein and C-terminal fragments of 30–56 kDa. These hydrophilic C-terminal fragments remained membrane-associated. A C-terminal proline-rich domain (PRD) was identified as the mediator of membrane association. The α1CPRD bound to SH3 domains in Src, Lyn, Hck, and the channel β2subunit. Mutant α1Csubunits lacking either ∼50 kDa of the C terminus or the PRD produced increased barium currents through the channels, demonstrating that these domains participate in the previously described (Wei, X., Neely, a., Lacerda, A. E. Olcese, r., Stefani, E., Perez-Reyes, E., and Birnbaumer, L. (1994) J. Biol. Chem.269, 1635–1640) inhibition of channel function by the C terminus.

In the present study, we investigated the role of channel subunits in the membrane targeting of voltage-dependent L-type calcium channel complexes. We co-expressed the calcium channel pore-forming α1Csubunit with different accessory β subunits in HEK-tsA201 cells and examined the subcellular localization of the channel subunits by immunohistochemistry using confocal microscopy and whole-cell radioligand binding studies. While the pore-forming α1Csubunit exhibited perinuclear staining when expressed alone, and several of the wild-type and mutant β subunits also exhibited intracellular staining, co-expression of the α1Csubunit with either the wild-type β2asubunit, a palmitoylation-deficient β2a(C3S/C4S) mutant or three other nonpalmitoylated β isoforms (β1b, β3, and β4subunits) resulted in the redistribution of both the α1Cand β subunits into clusters along the cell surface. Furthermore, the redistribution of calcium channel complexes to the plasma membrane was observed when α1Cwas co-expressed with an N- and C-terminal truncated mutant β2acontaining only the central conserved regions. However, when the α1Csubunit was co-expressed with an α1β interaction-deficient mutant, β2aBID−, we did not observe formation of the channels at the plasma membrane. In addition, an Src homology 3 motif mutant of β2athat was unable to interact with the α1Csubunit also failed to target channel complexes to the plasma membrane. Interestingly, co-expression of the pore-forming α1Csubunit with the largely peripheral accessory α2δ subunit was ineffective in recruiting α1Cto the plasma membrane, while co-distribution of all three subunits was observed when β2awas co-expressed with the α1Cand α2δ subunits. Taken together, our results suggested that the signal necessary for correct plasma membrane targeting of the class C L-type calcium channel complexes is generated as a result of a functional interaction between the α1and β subunits.

The properties of cardiac L-type channels have been well characterized electrophysiologically, and many such studies have demonstrated that the channels are regulated by a cAMP-dependent pathway. However, the subunit composition of native cardiac L-type calcium channels has not been completely defined. Furthermore, a very important question exists regarding the status of the C-terminal domain of the pore-forming α1subunit, as this domain has the potential to be the target of protein kinases but may be truncated as a result of post-translational processing. In the present studies, the α1Cand β2subunits were identified by subunit-specific antibodies after partial purification from heart membranes, or immunoprecipitation from cardiac myocytes. Both the β2and the full-length α1Csubunits were found to be expressed and co-localized in intact cardiac myocytes along T-tubule membranes. Using a quantitative antibody binding analysis, we demonstrated that the majority of the α1Csubunits in intact cardiac myocytes appear to be full-length. In addition, we observed that adenylyl cyclase is localized in a pattern similar to the channel subunits in cardiac myocytes. Taken together, our results provide new insights into the structural basis for understanding the regulation of L-type calcium channels by a cAMP-mediated signaling pathway.

In this study, we report that palmitoylation was a critical determinant of the subcellular localization of the rat β2asubunit of voltage-dependent calcium channels. Immunohistochemical staining of transfected cells revealed that a palmitoylation-deficient β2asubunit exhibited a diffuse intracellular staining pattern, in contrast to the plasma membrane distribution seen with the wild-type β2asubunit. Unexpectedly, mutations in regions distal to the palmitoylation sites at Cys3and Cys4affected palmitoylation of the β2aprotein. Mutations in ansrchomology 3 motif of the β2asubunit affected both palmitoylation and subcellular localization of the β2aprotein. A mutation in the β interaction domain, which disrupted interactions between the expressed α1and β subunits, also resulted in a decreased palmitoylation and diffuse intracellular localization of the β2aprotein. Studies of chimeric proteins revealed that the 16-amino acid N terminus of the β2asubunit was sufficient to confer palmitoylation to the nonpalmitoylated β1band β3isoforms. However, palmitoylation of chimeric β subunits was by itself insufficient to restore the plasma membrane localization observed with the wild-type β2aprotein. Treatment of transfected cells with brefeldin A increased the amount of palmitic acid incorporated in the β2aprotein, suggesting that palmitoylation of β2aoccurs during or shortly after protein synthesis. Two other β2variants, the rabbit β2aand β2b, which lack the palmitoylation sties at Cys3and Cys4, exhibited a diffuse intracellular staining pattern and were not palmitoylated.