Ciliary proteins Fap43 and Fap44 interact with each other and are essential for proper cilia and flagella beating

Cilia beating is powered by the inner and outer dynein arms (IDAs and ODAs). These multi-subunit macrocomplexes are arranged in two rows on each outer doublet along the entire cilium length, except its distal end. To generate cilia beating, the activity of ODAs and IDAs must be strictly regulated locally by interactions with the dynein arm-associated structures within each ciliary unit and coordinated globally in time and space between doublets and along the axoneme. Here, we provide evidence of a novel ciliary complex composed of two conserved WD-repeat proteins, Fap43p and Fap44p. This complex is adjacent to another WD-repeat protein, Fap57p, and most likely the two-headed inner dynein arm, IDA I1. Loss of either protein results in altered waveform, beat stroke and reduced swimming speed. The ciliary localization of Fap43p and Fap44p is interdependent in the ciliate Tetrahymena thermophila.     

Parvalbumin alters mitochondrial dynamics and affects cell morphology

The Ca²⁺-binding protein parvalbumin (PV) and mitochondria play important roles in Ca²⁺ signaling, buffering and sequestration. Antagonistic regulation of PV and mitochondrial volume is observed in in vitro and in vivo model systems. Changes in mitochondrial morphology, mitochondrial volume and dynamics (fusion, fission, mitophagy) resulting from modulation of PV were investigated in MDCK epithelial cells with stable overexpression/downregulation of PV. Increased PV levels resulted in smaller, roundish cells and shorter mitochondria, the latter phenomenon related to reduced fusion rates and decreased expression of genes involved in mitochondrial fusion. PV-overexpressing cells displayed increased mitophagy, a likely cause for the decreased mitochondrial volumes and the smaller overall cell size. Cells showed lower mobility in vitro, paralleled by reduced protrusions. Constitutive PV down-regulation in PV-overexpressing cells reverted mitochondrial morphology and fractional volume to the state present in control MDCK cells, resulting from increased mitochondrial movement and augmented fusion rates. PV-modulated, bi-directional and reversible mitochondrial dynamics are key to regulation of mitochondrial volume.     

Platelet-derived growth factor receptor beta identifies mesenchymal stem cells with enhanced engraftment to tissue injury and pro-angiogenic property

Mesenchymal stem cells (MSCs) are heterogeneous likely consisting of subpopulations with various therapeutic potentials. Here we attempted to acquire a subset of MSCs with enhanced effect in wound healing. We found that human placental MSCs expressing platelet-derived growth factor (PDGF) receptor (PDGFR)-β exhibited greater proliferation rates and generated more colony-forming unit-fibroblast (CFU-F), compared to PDGFR-β^? MSCs. Notably, PDGFR-β⁺ MSCs expressed higher levels of pro-angiogenic factors such as Ang1, Ang2, VEGF, bFGF and PDGF. When 10⁶ GFP-expressing MSCs were topically applied into excisional wounds in mice, PDGFR-β⁺ MSCs actively incorporated into the wound tissue, resulting in enhanced engraftment (3.92 ± 0.31 × 10⁵ remained in wound by 7 days) and accelerated wound closure; meanwhile, PDGFR-β^? MSCs tended to remain on the top of the wound bed with significantly fewer cells (2.46 ± 0.26 × 10⁵) engrafted into the wound, suggesting enhanced chemotactic migration and engraftment of PDGFR-β⁺ MSCs into the wound. Real-Time PCR and immunostain analyses revealed that the expression of PDGF-B was upregulated after wounding; transwell migration assay showed that PDGFR-β⁺ MSCs migrated eightfold more than PDGFR-β^? MSCs toward PDGF-BB. Intriguingly, PDGFR-β⁺ MSC-treated wounds showed significantly enhanced angiogenesis compared to PDGFR-β^? MSC- or vehicle-treated wounds. Thus, our results indicate that PDGFR-β identifies a subset of MSCs with enhanced chemotactic migration to wound injury and effect in promoting angiogenesis and wound healing, implying a greater therapeutic potential for certain diseases.     

Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M

The protein kinase D (PKD) family of proteins are important regulators of tumor growth, development, and progression. CRT0066101, an inhibitor of PKD, has antitumor activity in multiple types of carcinomas. However, the effect and mechanism of CRT0066101 in bladder cancer are not understood. In the present study, we show that CRT0066101 suppressed the proliferation and migration of four bladder cancer cell lines in vitro. We also demonstrate that CRT0066101 blocked tumor growth in a mouse flank xenograft model of bladder cancer. To further assess the role of PKD in bladder carcinoma, we examined the three PKD isoforms and found that PKD2 was highly expressed in eight bladder cancer cell lines and in urothelial carcinoma tissues from the TCGA database, and that short hairpin RNA (shRNA)-mediated knockdown of PKD2 dramatically reduced bladder cancer growth and invasion in vitro and in vivo, suggesting that the effect of the compound in bladder cancer is mediated through inhibition of PKD2. This notion was corroborated by demonstrating that the levels of phospho-PKD2 were markedly decreased in CRT0066101-treated bladder tumor explants. Furthermore, our cell cycle analysis by flow cytometry revealed that CRT0066101 treatment or PKD2 silencing arrested bladder cancer cells at the G2/M phase, the arrest being accompanied by decreases in the levels of cyclin B1, CDK1 and phospho-CDK1 (Thr161) and increases in the levels of p27^Kip1 and phospho-CDK1 (Thr14/Tyr15). Moreover, CRT0066101 downregulated the expression of Cdc25C, which dephosphorylates/activates CDK1, but enhanced the activity of the checkpoint kinase Chk1, which inhibits CDK1 by phosphorylating/inactivating Cdc25C. Finally, CRT0066101 was found to elevate the levels of Myt1, Wee1, phospho-Cdc25C (Ser216), Gadd45α, and 14-3-3 proteins, all of which reduce the CDK1-cyclin B1 complex activity. These novel findings suggest that CRT0066101 suppresses bladder cancer growth by inhibiting PKD2 through induction of G2/M cell cycle arrest, leading to the blockade of cell cycle progression.     

Macropinocytosis,mTORC1 and cellular growth control

The growth and proliferation of metazoan cells are driven by cellular nutrient status and by extracellular growth factors. Growth factor receptors on cell surfaces initiate biochemical signals that increase anabolic metabolism and macropinocytosis, an actin-dependent endocytic process in which relatively large volumes of extracellular solutes and nutrients are internalized and delivered efficiently into lysosomes. Macropinocytosis is prominent in many kinds of cancer cells, and supports the growth of cells transformed by oncogenic K-Ras. Growth factor receptor signaling and the overall metabolic status of the cell are coordinated in the cytoplasm by the mechanistic target-of-rapamycin complex-1 (mTORC1), which positively regulates protein synthesis and negatively regulates molecular salvage pathways such as autophagy. mTORC1 is activated by two distinct Ras-related small GTPases, Rag and Rheb, which associate with lysosomal membranes inside the cell. Rag recruits mTORC1 to the lysosomal surface where Rheb directly binds to and activates mTORC1. Rag is activated by both lysosomal luminal and cytosolic amino acids; Rheb activation requires phosphoinositide 3-kinase, Akt, and the tuberous sclerosis complex-1/2. Signals for activation of Rag and Rheb converge at the lysosomal membrane, and several lines of evidence support the idea that growth factor-dependent endocytosis facilitates amino acid transfer into the lysosome leading to the activation of Rag. This review summarizes evidence that growth factor-stimulated macropinocytosis is essential for amino acid-dependent activation of mTORC1, and that increased solute accumulation by macropinocytosis in transformed cells supports unchecked cell growth.     

The TET enzymes

During the past decade, we have learnt that the most common DNA modification, 5-methylcytosine (5mC), playing crucial roles in development and disease, is not stable but can be actively reversed to its unmodified form via enzymatic catalysis involving the TET enzymes. These ground-breaking discoveries have been achieved thanks to technological advances in the detection of the oxidized forms of 5mC and to the boldness of individual scientists. The TET enzymes require molecular oxygen for their catalysis, making them important targets for hypoxia research. They also require special cofactors which enable additional levels of regulation. Moreover, mutations and other genetic alterations in TETs are found, especially in myeloid malignances. This review focuses on the kinetic and inhibitory properties of the TET enzymes and the role of TETs in cellular differentiation and transformation and in cancer.