ICK is essential for cell type‐specific ciliogenesis and the regulation of ciliary transport

Cilia and flagella are formed and maintained by intraflagellar transport (IFT) and play important roles in sensing and moving across species. At the distal tip of the cilia/flagella, IFT complexes turn around to switch from anterograde to retrograde transport; however, the underlying regulatory mechanism is unclear. Here, we identified ICK localization at the tip of cilia as a regulator of ciliary transport. In ICK‐deficient mice, we found ciliary defects in neuronal progenitor cells with Hedgehog signal defects. ICK‐deficient cells formed cilia with mislocalized Hedgehog signaling components. Loss of ICK caused the accumulation of IFT‐A, IFT‐B, and BBSome components at the ciliary tips. In contrast, overexpression of ICK induced the strong accumulation of IFT‐B, but not IFT‐A or BBSome components at ciliary tips. In addition, ICK directly phosphorylated Kif3a, while inhibition of this Kif3a phosphorylation affected ciliary formation. Our results suggest that ICK is a Kif3a kinase and essential for proper ciliogenesis in development by regulating ciliary transport at the tip of cilia.     

Identification of cell type–specific correlations between ERK activity and cell viability upon treatment with ERK1/2 inhibitors

Increased MAPK signaling is a hallmark of various cancers and is a central regulator of cell survival. Direct ERK1/2 inhibition is considered a promising approach to avoid ERK1/2 reactivation caused by upstream kinases BRAF, MEK1/2, and KRAS, as well as by receptor tyrosine kinase inhibitors, but the dynamics and selectivity of ERK1/2 inhibitors are much less studied compared with BRAF or MEK inhibitors. Using ERK1/2 and downstream kinase ELK1 reporter cell lines of lung cancer (H1299; NRAS^Q61K), colon cancer (HCT-116; KRAS^G13D), neuroblastoma (SH-SY5Y), and leukemia (U937), we examined the relationship between ERK inhibition and drug-induced toxicity for five ERK inhibitors: SCH772984, ravoxertinib, LY3214996, ulixertinib, and VX-11e, as well as one MEK inhibitor, PD0325901. Comparing cell viability and ERK inhibition revealed different ERK dependencies for these cell lines. We identify several drugs, such as SCH772984 and VX-11e, which induce excessive toxicity not directly related to ERK1/2 inhibition in specific cell lines. We also show that PD0325901, LY3214996, and ulixertinib are prone to ERK1/2 reactivation over time. We distinguished two types of ERK1/2 reactivation: the first could be reversed by adding a fresh dose of inhibitors, while the second persists even after additional treatments. We also showed that cells that became resistant to the MEK1/2 inhibitor PD0325901 due to ERK1/2 reactivation remained sensitive to ERK1/2 inhibitor ulixertinib. Our data indicate that correlation of ERK inhibition with drug-induced toxicity in multiple cell lines may help to find more selective and effective ERK1/2 inhibitors.     

Cytokeratin 18 is a specific marker of bovine intestinal M cell

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches have an important role in mucosal immune responses. A primary difficulty for investigations of bovine M cells is the lack of a specific molecular marker. To identify such a marker, we investigated the expression of several kinds of intermediate filament proteins using calf Peyer's patches. The expression patterns of cytokeratin (CK) 18 in jejunal and ileal FAE were very similar to the localization pattern of M cells recognized by scanning electron microscopy. Mirror sections revealed that jejunal CK18-positive cells had irregular and sparse microvilli, as well as pocket-like structures containing lymphocytes, typical morphological characteristic of M cells. However, CK18-negative cells had regular and dense microvilli on their surface, typical of the morphology of enterocytes. In contrast, CK20 immunoreactivity was detected in almost all villous epithelial cells and CK18-negative cells in the FAE. CK18-positive proliferating transit-amplifying cells in the crypt exchanged CK18 for CK20 above the mouth of the crypt and after moving to the villi; however, CK18-positive M cells in the crypt continued their expression of CK18 during movement to the FAE region. Terminal deoxynucleotidyl-transferase-mediated deoxyuridine-triphosphate-biotin nick-end labeling-positive apoptotic cells were specifically detected at the apical region of villi and FAE in the jejunum and ileum, and all were also stained for CK20. These data indicate that CK18 may be a molecular marker for bovine M cells in FAE and that M cells may transdifferentiate to CK20-positive enterocytes and die by apoptosis in the apex of the FAE.     

Cell type–specific variations in the induction of hsp70 in human leukocytes by feverlike whole body hyperthermia

Fever has been associated with shortened duration and improved survival in infectious disease. The mechanism of this beneficial response is still poorly understood. The heat-inducible 70-kDa heat shock protein (Hsp70) has been associated with protection of leukocytes against the cytotoxicity of inflammatory mediators and with improved survival of severe infections. This study characterizes the induction of Hsp70 by feverlike temperatures in human leukocytes in vitro and in vivo. Using flow cytometry, Hsp70 expression was determined in whole blood samples. This approach eliminated cell isolation procedures that would greatly affect the results. Heat treatment of whole blood in vitro for 2 hours at different temperatures revealed that Hsp70 expression depends on temperature and cell type; up to 41 degrees C, Hsp70 increased only slightly in lymphocytes and polymorphonuclear leukocytes. However, in monocytes a strong induction was already seen at 39 degrees C, and Hsp70 levels at 41 degrees C were 10-fold higher than in the 37 degrees C control. To be as close as possible to the physiological situation during fever, we immersed healthy volunteers in a hot water bath, inducing whole body hyperthermia (39 degrees C), and measured leukocyte Hsp70 expression. Hsp70 was induced in all leukocytes with comparable but less pronounced cell type-specific variations as observed in vitro. Thus, a systemic increase of body temperature as triggered by fever stimulates Hsp70 expression in peripheral leukocytes, especially in monocytes. This fever-induced Hsp70 expression may protect monocytes when confronted with cytotoxic inflammatory mediators, thereby improving the course of the disease.     

Positioning and stability of mitotic spindle orientation in the neuroepithelial cell

To verify non-random positioning and to define the stability of the mitotic spindle orientation in neuroepithelial cells of mouse foetuses, computer - assisted morphometric analysis at the light microscopy level was performed. It was confirmed that the mitotic spindle axis is positioned non-randomly in relation to the cell polarity axis and could be displaced only within a narrow range. This orientation was found to be attained at metaphase and it does not change until telophase is completed. However, in relation to the long axis of the neural tube the mitotic spindle axis was found to be positioned randomly. In the light of these findings centrosome movement and positioning are discussed.     

Reporter mice for isolating and auditing cell type‐specific extracellular vesicles in vivo

Extracellular vesicles (EVs) are abundant, lipid‐enclosed vectors that contain nucleic acids and proteins, they can be secreted from donor cells and freely circulate, and they can be engulfed by recipient cells thus enabling systemic communication between heterotypic cell types. However, genetic tools for labeling, isolating, and auditing cell type‐specific EVs in vivo, without prior in vitro manipulation, are lacking. We have used CRISPR‐Cas9‐mediated genome editing to generate mice bearing a CD63‐emGFP^{loxP/stop/loxP} knock‐in cassette that enables the specific labeling of circulating CD63⁺ vesicles from any cell type when crossed with lineage‐specific Cre recombinase driver mice. As proof‐of‐principle, we have crossed these mice with Cdh5‐Cre^ERT2 mice to generate CD63^emGFP+ vasculature. Using these mice, we show that developing vasculature is marked with emerald GFP (emGFP) following tamoxifen administration to pregnant females. In adult mice, quiescent vasculature and angiogenic vasculature (in tumors) is also marked with emGFP. Moreover, whole plasma‐purified EVs contain a subpopulation of emGFP⁺ vesicles that are derived from the endothelium, co‐express additional EV (e.g., CD9 and CD81) and endothelial cell (e.g., CD105) markers, and they harbor specific miRNAs (e.g., miR‐126, miR‐30c, and miR‐125b). This new mouse strain should be a useful genetic tool for generating cell type‐specific, CD63⁺ EVs that freely circulate in serum and can subsequently be isolated and characterized using standard methodologies.     

Oncoprotein 18 is a phosphorylation-responsive regulator of microtubule dynamics.

Oncoprotein 18 (Op18, also termed p19, p18, prosolin or stathmin) is a cytosolic protein of previously unknown function. Phosphorylation of Op18 is cell cycle regulated by cyclin-dependent kinases (CDKs), and expression of a 'CDK target site-deficient mutant' results in a phenotype indicative of a role for Op18 during mitosis. This phenotype is compatible with the idea that Op18 is a phosphorylation-responsive regulator of microtubule (MT) dynamics. Therefore, in this study, we analyzed MTs in cells induced to express either wild-type or mutated Op18. The results showed that wild-type Op18 and a CDK target site mutant both efficiently elicited rapid depolymerization of MTs. This result contrasts with clear-cut differences in their cell cycle phenotypes. Morphological analysis of MTs explained this apparent discrepancy: while interphase MTs were depolymerized in cells expressing either Op18 derivative, apparently normal mitotic spindles were formed only in cells overexpressing wild-type Op18. This result correlates with our finding that only mutated Op18 causes a block during mitosis. Hence, we conclude that Op18 decreases MT stability and that this activity of Op18 is subject to cell cycle regulation by CDKs.     

Roles and mechanisms of Kinesin-6 KIF20A in spindle organization during cell division

Mitotic kinesin is crucial for spindle assembly and chromosome segregation in cell division. KIF20A/MKlp2, a member of kinesin-6 subfamily, plays important roles in the central spindle organization at anaphase and cytokinesis. In this review, we briefly introduce the discovery and classification of kinesin-6 motors in model organisms, and summarize the biochemical features and mechanics of KIF20A proteins. We emphasize the complicated interactions of KIF20A with partner proteins, including MKlp1, Plk1 and Rab6. Particularly, we highlight the regulation of Cdk1 and chromosomal passenger complex on kinesin-6 KIF20A at late stage of mitosis. We summarized the multiple functions of KIF20A in central spindle assembly and the formation of cleavage furrow in both mitosis and meiosis. In addition, we conclude the expression patterns of KIF20A in tumorigenesis and its applications in tumor therapy.     

Prohibitin is a cholesterol‐sensitive regulator of cell cycle transit

Cholesterol is essential in establishing most functional animal cell membranes; cells cannot grow or proliferate in the absence of sufficient cholesterol. Consequently, almost every cell, tissue, and animal tightly regulates cholesterol homeostasis, including complex mechanisms of synthesis, transport, uptake, and disposition of cholesterol molecules. We hypothesize that cellular recognition of cholesterol insufficiency causes cell cycle arrest in order to avoid a catastrophic failure in membrane synthesis. Here, we demonstrate using unbiased proteomics and standard biochemistry that cholesterol insufficiency causes upregulation of prohibitin, an inhibitor of cell cycle progression, through activation of a cholesterol‐responsive promoter element. We also demonstrate that prohibitin protects cells from apoptosis caused by cholesterol insufficiency. This is the first study tying cholesterol homeostasis to a specific cell cycle regulator that inhibits apoptosis. J. Cell. Biochem. 111: 1367–1374, 2010. © 2010 Wiley‐Liss, Inc.     

Human epidermis is a novel site of phospholipase B expression

Phospholipase B (PLB) is an enzyme that displays both phospholipase A(2) and lysophospholipase activities. Analysis of human epidermis homogenates indicated the presence of a 97 kDa PLB protein, as well as a phospholipase A(2) activity, both being enriched in the soluble fraction. Immunolabelling and in situ hybridization experiments showed that this enzyme is expressed in the different layers of epidermis with an accumulation at the dermo-epidermis junction. RT-PCR data indicated that PLB is specifically expressed in natural and reconstructed epidermis. By 3'-RACE-PCR and screening of human genome databases, we obtained a 3600 bp cDNA coding for human PLB highly homologous to already described intestinal brush border PLBs. These data led us to conclude that the soluble PLB corresponds to a proteolytic cleavage of the membrane anchored protein. Altogether, our results provide the first characterization of human PLB which should play an important role in epidermal barrier function.     

Mammalian inscuteable regulates spindle orientation and cell fate in the developing retina

During mammalian neurogenesis, progenitor cells can divide with the mitotic spindle oriented parallel or perpendicular to the surface of the neuroepithelium. Perpendicular divisions are more likely to be asymmetric and generate one progenitor and one neuronal precursor. Whether the orientation of the mitotic spindle actually determines their asymmetric outcome is unclear. Here, we characterize a mammalian homolog of Inscuteable (mInsc), a key regulator of spindle orientation in Drosophila. mInsc is expressed temporally and spatially in a manner that suggests a role in orienting the mitotic spindle in the developing nervous system. Using retroviral RNAi in rat retinal explants, we show that downregulation of mInsc inhibits vertical divisions. This results in enhanced proliferation, consistent with a higher frequency of symmetric divisions generating two proliferating cells. Our results suggest that the orientation of neural progenitor divisions is important for cell fate specification in the retina and determines their symmetric or asymmetric outcome.     

Protein abundance of AKT and ERK pathway components governs cell type‐specific regulation of proliferation

Signaling through the AKT and ERK pathways controls cell proliferation. However, the integrated regulation of this multistep process, involving signal processing, cell growth and cell cycle progression, is poorly understood. Here, we study different hematopoietic cell types, in which AKT and ERK signaling is triggered by erythropoietin (Epo). Although these cell types share the molecular network topology for pro‐proliferative Epo signaling, they exhibit distinct proliferative responses. Iterating quantitative experiments and mathematical modeling, we identify two molecular sources for cell type‐specific proliferation. First, cell type‐specific protein abundance patterns cause differential signal flow along the AKT and ERK pathways. Second, downstream regulators of both pathways have differential effects on proliferation, suggesting that protein synthesis is rate‐limiting for faster cycling cells while slower cell cycles are controlled at the G1‐S progression. The integrated mathematical model of Epo‐driven proliferation explains cell type‐specific effects of targeted AKT and ERK inhibitors and faithfully predicts, based on the protein abundance, anti‐proliferative effects of inhibitors in primary human erythroid progenitor cells. Our findings suggest that the effectiveness of targeted cancer therapy might become predictable from protein abundance.