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Abstract Cellular senescence occurs through the accumulation of many kinds of stresses. Senescent cells in tissues also cause various age-related disorders. Therefore, detecting them without labeling is beneficial for medical research and developing diagnostic methods. However, existing biomarkers have limitations of requiring fixation and labeling, or their molecular backgrounds are uncertain. Coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging is a novel option because it can assess and visualize molecular structures based on their molecular fingerprint. Here, we present a new label-free method to visualize cellular senescence using CARS imaging in nucleoli. We found the peak of the nucleolar amide I band shifted to a higher wavenumber in binuclear senescent cells, which reflects changes in the protein secondary structure from predominant α-helices to β-sheets originating from amyloid-like aggregates. Following this, we developed a procedure that can visualize the senescent cells by providing the ratios and subtractions of these two components. We also confirmed that the procedure can visualize nucleolar aggregates due to unfolded/misfolded proteins produced by proteasome inhibition. Finally, we found that this method can help visualize the nucleolar defects in naïve cells even before binucleation. Thus, our method is beneficial to evaluate ongoing cellular senescence through label-free imaging of nucleolar defects.

Abstract Malignant mesothelioma (MMe) is a rare but aggressive malignancy. Although the molecular genetics of MMe is known, including BRCA1-associated protein-1 (BAP1) gene alterations, the prognosis of MMe patients remains poor. Here, we generated BAP1 knockout (BAP1-KO) human mesothelial cell clones to develop molecular-targeted therapeutics based on genetic alterations in MMe. cDNA microarray and quantitative RT-PCR (qRT-PCR) analyses revealed high expression of a calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D) gene in the BAP1-KO cells. CAMK2D was highly expressed in 70% of the human MMe tissues (56/80) and correlated with the loss of BAP1 expression, making it a potential diagnostic and therapeutic target for BAP1-deficient MMe. We screened an anticancer drugs library using BAP1-KO cells and successfully identified a CaMKII inhibitor, KN-93, which displayed a more potent and selective antiproliferative effect against BAP1-deficient cells than cisplatin or pemetrexed. KN-93 significantly suppressed the tumor growth in mice xenografted with BAP1-deficient MMe cells. This study is the first to provide a potential molecular-targeted therapeutic approach for BAP1-deficient MMe.

We analyzed the effects of a hypo-osmotic environment on rat Schwann cells, a type of glial cell surrounding neurons, using ultra-broadband multiplex coherent anti-Stokes Raman scattering (CARS) microscopy. After hypo-osmotic treatment, we detected vacuole-like components in the cytoplasm using both bright-field and CARS spectroscopic imaging. An approach integrating both morphological examination and a spectroscopic analysis based on multiple vibrational bands revealed that these structures are predominantly water-filled, and their characteristics closely resembled those of the vacuoles observed in plant cells.

Previous genome-wide association studies have identified risk loci for pancreatic cancer but were centered on individuals of European ancestry. Here the authors identify GP2 gene variants associated with pancreatic cancer susceptibility in populations of East Asian ancestry.

We have developed an ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopic system using a supercontinuum (SC) seeded by sub-100-ps (85 ps) laser pulses with a sub-MHz (0.82 MHz) repetition rate. Because of the high peak power and ultrabroadband spectral profile of the SC, we can efficiently generate multiplex CARS signals in the spectral range of 600–3600 cm−1, which covers the entire molecular fingerprint region, as well as the C—H and O—H stretching regions. Due to the high peak power of the new laser source, the exposure time (pixel dwell time) for CARS imaging of polymer beads was reduced to less than 1 ms (0.8 ms), which was limited by the readout time of a CCD camera. Owing to the improvement in CARS spectral quality, clear molecular fingerprinting was achieved for living HeLa cells at different phases in the cell cycle.

Keratins (Ks), the intermediate filament (IF) proteins of epithelia, are coordinately expressed as pairs in a cell-lineage and differentiation manner. Cortical thymic epithelial cells (cTECs) predominantly express the simple epithelium keratin 8/18 (K8/K18) pair, whereas medullary thymic epithelial cells (mTECs) express the stratified epithelium K5/K14 pair, with TECs exhibiting K5 and K8 at the cortico-medullary junction in mature thymus. In the work reported here, we used wild-type (WT) and K8-knockout (K8-null) mice to address the contribution of K8/K18 IFs in the maintenance of the thymic epithelial structure. K8-null thymus maintained the differential cell segregation at the cortex versus the medulla observed in WT thymus, and the distribution of immature thymocytes at the cortex. The K8/K18 loss did not affect thymocyte development. However, it massively perturbed the TEC morphology both at the cortex and the medulla, along with a prominent depletion of cTECs. Such tissue alterations coincided with an increase in apoptosis and a reduced expression of Albatross (Fas-binding factor-1), also known for its capacity to bind K8/18 IFs. In addition, the K8/K18 loss affected the distribution of K5/K14-positive mTECs, but not their differentiation status. Together, the results indicate that K8/K18 IFs constitute key promoters of the thymic epithelium integrity.

Keratins 8 and 18 (collectively referred to as K8/K18) are the major components of intermediate filaments of simple epithelial cells. Recent studies have revealed the function of K8/K18 in apoptosis modulation. Here, we show that eIF3k, originally identified as the smallest subunit of eukaryotic translation initiation factor 3 (eIF3) complexes, also localizes to keratin intermediate filaments and physically associates with K18 in epithelial cells. Upon induction of apoptosis, eIF3k colocalizes with K8/K18 in the insoluble cytoplasmic inclusions. Depletion of endogenous eIF3k de-sensitizes simple epithelial cells to various types of apoptosis through a K8/K18-dependent mechanism and promotes the retention of active caspase 3 in cytoplasmic inclusions by increasing its binding to keratins. Consequently, the cleavage of caspase cytosolic and nuclear substrates, such as ICAD and PARP, respectively, is reduced in eIF3k-depleted cells. This study not only reveals the existence of eIF3k in a subcellular compartment other than the eIF3 complex, but also identifies an apoptosis-promoting function of eIF3k in simple epithelial cells by relieving the caspase-sequestration effect of K8/K18, thereby increasing the availability of caspases to their non-keratin-residing substrates. [ABSTRACT FROM AUTHOR]

Tetraploidy, a state in which cells have doubled chromosomal sets, is observed in ~20% of solid tumors and is considered to frequently precede aneuploidy in carcinogenesis. Tetraploidy is also detected during terminal differentiation and represents a hallmark of aging. Most tetraploid cultured cells are arrested by p53 stabilization. However, the fate of tetraploid cells in vivo remains largely unknown. Here, we analyze the ability to repair wounds in the skin of phosphovimentin-deficient (VIMSA/SA) mice. Early into wound healing, subcutaneous fibroblasts failed to undergo cytokinesis, resulting in binucleate tetraploidy. Accordingly, the mRNA level of p21 (a p53-responsive gene) was elevated in a VIMSA/SA-specific manner. Disappearance of tetraploidy coincided with an increase in aneuploidy. Thereafter, senescence-related markers were significantly elevated in VIMSA/SA mice. Because our tetraploidy-prone mouse model also exhibited subcutaneous fat loss at the age of 14 months, another premature aging phenotype, our data suggest that following cytokinetic failure, a subset of tetraploid cells enters a new cell cycle and develops into aneuploid cells in vivo, which promote premature aging. [ABSTRACT FROM AUTHOR]

Vimentin, a type III intermediate filament (IF) protein, is phosphorylated predominantly in mitosis. The expression of a phosphorylation-compromised vimentin mutant in T24 cultured cells leads to cytokinetic failure, resulting in binucleation (multinucleation). The physiological significance of intermediate filament phosphorylation during mitosis for organogenesis and tissue homeostasis was uncertain. Here, we generated knock-in mice expressing vimentin that have had the serine sites phosphorylated during mitosis substituted by alanine residues. Homozygotic mice (VIMSA/SA) presented with microophthalmia and cataracts in the lens, whereas heterozygotic mice (VIMWT/SA) were indistinguishable from WT (VIMWT/WT) mice. In VIMSA/SA mice, lens epithelial cell number was not only reduced but the cells also exhibited chromosomal instability, including binucleation and aneuploidy. Electron microscopy revealed fiber membranes that were disorganized in the lenses of VIMSA/SA, reminiscent of similar characteristic changes seen in age-related cataracts. Because the mRNA level of the senescence (aging)-related gene was significantly elevated in samples from VIMSA/SA, the lens phenotype suggests a possible causal relationship between chromosomal instability and premature aging. [ABSTRACT FROM AUTHOR]

Primary cilia protrude from the surface of quiescent cells and disassemble at cell cycle reentry. We previously showed that ciliary reassembly is suppressed by trichoplein-mediated Aurora A activation pathway in growing cells. Here, we report that Ndel1, a well-known modulator of dynein activity, localizes at the subdistal appendage of the mother centriole, which nucleates a primary cilium. In the presence of serum, Ndel1 depletion reduces trichoplein at the mother centriole and induces unscheduled primary cilia formation, which is reverted by forced trichoplein expression or coknockdown of KCTD17 (an E3 ligase component protein for trichoplein). Serum starvation induced transient Ndel1 degradation, subsequent to the disappearance of trichoplein at the mother centriole. Forced expression of Ndel1 suppressed trichoplein degradation and axonemal microtubule extension during ciliogenesis, similar to trichoplein induction or KCTD17 knockdown. Most importantly, the proportion of ciliated and quiescent cells was increased in the kidney tubular epithelia of newborn Ndel1-hypomorphic mice. Thus, Ndel1 acts as a novel upstream regulator of the trichoplein--Aurora A pathway to inhibit primary cilia assembly. [ABSTRACT FROM AUTHOR]

Accumulating evidence points to cross-talk between Fcϵ RI and CC chemokine receptor (CCR)-mediated signaling pathways in mast cells. Here, we propose that vimentin, a protein comprising type III intermediate filament, participates in such cross-talk for CCL2/monocyte chemotactic protein 1 (MCP-1) production in mast cells, which is a mechanism for allergic inflammation. Co-stimulation via FcϵRI, using IgE/antigen, and CCR1, using recombinant CCL3/macrophage inflammatory protein-1α (MIP-1α), increased expression of phosphorylated, disassembled, and soluble vimentin in rat basophilic leukemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells) and bone marrow-derived murine mast cells, both models of mucosal type mast cells. Furthermore, co-stimulation enhanced production of CCL2 as well as phosphorylation of MAPK. Treating the cells with p38MAPKinhibitor SB203580, but not withMEK inhibitor PD98058, reduced CCL2 production, suggesting that p38 MAPK, but not ERK1/2, plays a critical role in the chemokine production. Immunoprecipitation analysis showed that vimentin interacts with phosphorylated ERK1/2 and p38 MAPKs in the co-simulated cells. Preventing disassembly of the vimentin by aggregating vimentin filaments using β,β'-iminodipropionitrile reduced the interaction of vimentin with phosphorylated MAPKs as well as CCL2 production in the cells. Taken together, disassembled vimentin interacting with phosphorylated p38 MAPK could mediate CCL2 production in mast cells upon FcϵRI and CCR1 activation. [ABSTRACT FROM AUTHOR]