BMC Molecular and Cell Biology

In culture system, environmental factors, such as increasing exogenous growth factors and adhesion to type I collagen (Col-I) induce epithelial-to-mesenchymal transition (EMT) in cells. Col-I molecules maintain a non-fibril form under acidic conditions, and they reassemble into fibrils under physiological conditions. Col-I fibrils often assemble to form three-dimensional gels. The gels and non-gel-form of Col-I can be utilized as culture substrates and different gel-forming state often elicit different cell behaviors. However, gel-form dependent effects on cell behaviors, including EMT induction, remain unclear. EMT induction in lung cancer cell line A549 has been reported via adhesion to Col-I but the effects of gel form dependency are unelucidated. This study investigated the changes in EMT-related behaviors in A549 cells cultured on Col-I gels. We examined cell morphology, proliferation, single-cell migration and expression of EMT-related features in A549 cells cultured on gels or non-gel form of Col-I and non-treated dish with or without transforming growth factor (TGF)-{beta}1. On Col-I gels, some cells kept cell-cell contacts and formed clusters, others maintained single-cell form. In cell-cell contact regions, E-cadherin expression was downregulated, whereas that of N-cadherin was upregulated. Vimentin and integrins 2 and {beta}1 expression were not increased. In TGF-{beta}1-treated A549 cells, cadherin switched from E- to N-cadherin. Their morphology changed to a mesenchymal form and cells scattered with no cluster formation. Vimentin, integrins 2 and {beta}1 expression were upregulated. Thus, we concluded that culture on Col-I fibrous gels induced E- to N-cadherin switching without other EMT-related phenotypes in A549 cells.

Mast cells are abundant in melanoma tumors, and studies suggest that they can be either detrimental or protective for melanoma growth. However, the underlying mechanisms are not fully understood. Here, we adopted a hanging drop-established spheroid system to investigate how mast cells can influence melanoma growth and phenotype in a 3-D context. In the presence of mast cells or mast cell-conditioned medium, melanoma spheroid growth was profoundly reduced. To address the underlying mechanism, we conducted a transcriptomic analysis, which revealed that mast cell-conditioned medium had extensive effects on the melanoma gene expression patterns. Pathway analyses revealed profound effects on the expression of genes related to amino acid and protein metabolism. The conditioned medium also induced an upregulated expression of cancer-related genes, including adhesion molecules implicated in metastatic spreading. In line with this, after transfer to a Matrigel extracellular matrix milieu, spheroids that had been developed in the presence of mast cell-conditioned medium displayed enhanced elevated growth and adhesive properties. However, when assessing for possible effects of nutrient starvation, i.e., reduced nutrient content in mast cell-conditioned medium, we found that the observed effects on melanoma spheroid growth potentially could be explained by such effects. Hence, it cannot be excluded that the observed phenotypic alterations of melanoma spheroids grown in the presence of mast cells or mast cell-conditioned media are, at least partly, due to nutrient starvation rather than to the action of factors secreted by mast cells. Instead, our findings may provide insight into the effects on gene expression events that occur in melanoma tumors under nutrient stress.

Cells in hybrid state of the epithelial-to-mesenchymal transition (EMT) have been shown to be responsible for tumor cell metastasis. However, the precise mechanisms underlying the morphological changes and acquisition of invasive phenotypes in hybrid EMT cells are still unknown. Here, we introduced the deletion of a proto-cadherin and well described oncogene, FAT1, in skin carcinoma cells to generate a hybrid state of EMT. Surprisingly, the FAT1 knock-out (KO) cells were less motile than the parental non-mutated cell line they were derived from. However, we observed that FAT1 KO cells secrete specific factors in the form of extra-cellular vesicles into their microenvironment, which promote the migration of surrounding non-mutant cells. When stimulated with these extracellular vesicles, groups of non-mutated parental cells collectively migrated faster and formed finger-like instabilities at the migrating front. Furthermore, we found that the actomyosin contractility of FAT1 KO cells in hybrid EMT states was much lower than the parental cells. It appeared that the factors secreted by FAT1 KO cells relaxed the traction forces in recipient cells. This force release likely fostered the scattering and migration of non-mutated cells surrounding FAT1 mutant cells. Thus, we characterized a non-autonomous promotion of cell invasiveness in the cancer cells surrounding FAT1-deficient cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/556588v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@104063eorg.highwire.dtl.DTLVardef@135f9fforg.highwire.dtl.DTLVardef@b0052eorg.highwire.dtl.DTLVardef@243864_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure:C_FLOATNO Schematic showing how FAT1 deletion stimulates the migration of neighboring non mutant cells. FAT1 KO cells secrete extracellular vesicles that carry factors that promote migration of non-mutant control cells, possibly through relaxation of traction forces in the recipient cells. C_FIG

The cohesin protein complex plays a very important role in chromosome segregation, transcription, DNA replication and chromosome condensation. Mutations in cohesin proteins give rise to a disease collectively referred to as Cohesinopathies. The major cause of Cohesinopathies arise due to defects associated with gene expression, that give rise to developmental disorders. In this study, we have used Saccharomyces cerevisiae to mimic the Cohesinopathy disorder Roberts syndrome with mutations (eco1W216G) homologous to that of humans (esco2). Our data suggests that polyol sugars like sorbitol, can repair misfolded proteins and reduce ER and proteostatic stress. We have used sorbitol as a chemical chaperone, to check how it can restore chromosome segregation, gene expression, misregulation, protein misfolding, autophagy and translational defects in the cohesin mutant of the Roberts phenotype. In silico screening has helped us identify the possible sites on eco1, which could be possibly altering the phenotypic traits. Article HighlightsIn presence of sorbitol O_LITemperature sensitivity of the Roberts mutants is rescued. C_LIO_LIChromosome cohesion defect is reduced. C_LIO_LITranslational defects are minimized. C_LIO_LIAutophagy is enhanced C_LIO_LIMolecular docking shows its interaction with the acetyltransferase domain of ESCO2. C_LI

Malignant melanoma is the most lethal form of skin cancer; its incidence has increased over the last five decades. Y-box binding protein 1 (YB-1) plays a prominent role in mediating metastatic behavior by promoting epithelial-to-mesenchymal transition (EMT) processes. Migratory melanoma cells exhibit two major phenotypes: elongated mesenchymal or rounded amoeboid. The actomyosin cytoskeleton is key in both phenotypes, but intermediate filaments also undergo a significant rearrangement process, switching from cytokeratin-rich to vimentin and nestin-rich network. In this study, we aimed to investigate to what extent YB-1 impacts the biomechanical (cell stiffness) and biochemical aspects of melanoma cells and their cytoskeleton. To this end, we subjected A375 YB-1 knock-out and parental cells to atomic force microscopy investigations (stiffness determination), immunolabelling, and proteome analysis. We found that YB-1 expressing cells were significantly stiffer compared to the corresponding YB-1 knock-out cell line. Proteomic analysis revealed that expression of YB-1 results in a strong co-expression of nestin, vimentin, fascin-1, and septin-9. In the YB-1 knock-out nestin was completely depleted, but zyxin was strongly upregulated. Collectively, our results showed that YB-1 knock-out acquires some characteristics of mesenchymal phenotype but lacks important markers of malignancy and invasiveness such as nestin or vimentin. We posit that there is an association of YB-1 expression with an amoeboid phenotype, which would explain the increased migratory capacity.

To facilitate rapid changes in morphology without endangering cell integrity, each cell possesses a substantial amount of cell surface excess (CSE) that can be promptly deployed to cover cell extensions. CSE can be stored in different types of small surface projections such as filopodia, microvilli, and ridges, with rounded bleb-like projections being the most common and rapidly achieved form of storage. We demonstrate in this paper that cells migrating in 3D collagen use CSE to cover the developing protrusions. After retraction of a protrusion, the CSE this produces is stored over the cell body similar to the CSE produced by cell rounding. For the coordinated process of CSE storage and release, all cells should have specific mechanisms of regulation, and we hypothesize that microtubules (MT) play an important role in this mechanism. We show here that different effects of MT depolymerization on cell motility such as inhibiting mesenchymal motility and enhancing amoeboid, can be explained by the essential role of MT in CSE regulation and dynamics.

14-3-3 protein family binds and regulate hundred of serine/threonine phosphorylated proteins. Considered as redundant, ubiquitous and constantly expressed this protein family was treated as an accessory for many signaling systems. Here we studied the reversible inhibition by acetylation of its essential N-{varepsilon}-lysine 49/51 residue during the osteogenic differentiation of human adipose-derived stem cells (ASC). We found that during the differentiation of ASC the levels of 14-3-3 acK49/51 increase showing that inhibition of 14-3-3 is necessary for this process. Among the 7 paralogs of this family, the inhibition by this posttranslational modification occurs mostly on the paralog {beta} located specifically in the nucleus where 14-3-3 was described to binds to H3 histone and many transcription factors. Short hairpin RNA silencing of 14-3-3{beta} gene but not 14-3-3{gamma} increases significantly the osteogenic potential of the cells. These results show that specifically 14-3-3{beta} is a negative regulator of osteogenesis and its inhibition by acetylation on lysine 51 is the cellular mechanism to regulate it.

Monolayer cell culture, while useful for basic in vitro studies, are not physiologically relevant. Spheroids, on the other hand provide a more complex 3-dimensional (3D) structure which more resemble the in vivo tumour growth thereby allowing results obtained with those on proliferation, cell death, differentiation, metabolism, and various anti-tumour therapies to be more predictive of in vivo outcomes. However, the cost associated with their generation often involve expensive, plate, media, and growth supplements, which have limited their use for high throughput experiments. The protocol herein presents a novel and rapid generation for single spheroids of various cancer cell lines, U87 MG; SEBTA-027; SF188, brain cancer cells, DU-145, TRAMP-C1, prostate cancer cells, in 96-round bottom well plates. Cells are washed with anti-adherent solution, and the homogeneous compact spheroid morphology was evidenced as early as 24 hours after 10 minutes centrifugation for the seeded cells. By using confocal microscopy, the proliferating cells were traced in the rim and the dead cells were found inside the core region of the spheroid. The H&E stain of spheroid slices and the western blotting were utilised to investigate the tightness of the cell packaging by adhesion proteins. Carnosine was used as an example of treatment for U87 single spheroids. The protocol allows the rapid generation of spheroids, which will help towards reducing the number of tests performed on animals.

Vaults are large cone-shaped and highly conservative ribonucleoprotein complexes present in the cells of most major eukaryote clades. However, despite their wide distribution, their functions and evolutionary dynamics still remain enigmatic. Several minor functions in modulating signaling cascades and multidrug resistance phenotypes were previously discovered for eukaryotic vaults, yet nothing is known about bacterial homologs of the major vault protein (MVP), a protein that comprises the entirety of vault external surface. Using gene and protein BLAST searches in NCBI and UniProt databases we identified a number of bacterial species in prokaryotic orders Myxococcales, Cytophagales and Oscillatoriales with >50% identity to eukaryotic MVP sequences. Interestingly, all of these species are characterized by one common feature - gliding type of motility. Secondary structures of the identified proteins were predicted using RAPTORX web service and aligned via jFATCAT-flexible algorithm in the RCSB PDB Java Structure Alignment tool to elucidate structural identity. Coiled coil domain at the MVP C-terminus of all studied bacterial species resembled TolA protein of Escherichia coli by both structure and sequence. We also showed that MVP sequences from chemotrophic bacteria Myxococcales and Cytophagales contain a domain homologous to eukaryotic band-7 domain, unlike cyanobacterial and eukaryotic major vault proteins. As expected, maximum-likelihood phylogenetic trees for MVP sequences separate studied taxa into two clades - first clade contains Oscillatoriales (Cyanobacteria) and Eukaryotes and the second one contains chemotrophic bacteria. In addition, binding prediction via RAPTORX showed great multiplicity GMP and CMP nucleoside monophosphate binding pockets in Myxococcales and Cytophagales MVP, unlike eukaryotic and cyanobacterial proteins which had much lower affinity to these substrates. Due to high similarity of eukaryotic and cyanobacterial MVP sequences and a pattern of its phylogenetic distribution, we can speculate that the most likely scenario for vault appearance in eukaryotes is horizontal gene transfer from cyanobacteria. Presence of GMP and CMP binding pockets in MVP could also point to a function in depleting cytosolic nucleotide concentration which would be beneficial, for instance, during a viral infection. Further research is necessary to uncover potential functions of this enigmatic protein in bacteria and to determine its evolutionary patterns. In addition, a correlation between MVP presence and gliding motility in bacteria could also lead to elucidating selective pressures on the early evolution of this protein.

Heterogeneity within a putative stem cell population presents a challenge for studies and applications of such cells. Cloning may provide a strategy for reducing heterogeneity. However, previous studies have the weakness in reliability of single-cell-origin of the colonies. The present study aims to apply an alternative method to obtain clonal dental pulp cells with increased reliability of single cell origin. Dental pulp cells were cultured from 13 human wisdom teeth. Primary cultures of 3 human benign tumors were included as comparison. Cells were seeded into wells of a 96-plate at a mean density of 1 cell/well. On the next day, wells were inspected one by one to identify wells with single cells which were followed for 3 weeks. Survived clones were expanded and further characterized. Single cells were observed in all cases, the number of single-cell-wells varied from 16 to 33. Three weeks later, survived and grown clonal cells were observed in 10 to 29 wells, giving surviving rates of 33-91%. By contrast, though single tumor cells were also observed, none of them survived. Expanded clones exhibited diversity in viability and osteogenic differentiation which also differed from their parental cells. Seeding cells at clonal density into physically separated compartments like wells in a 96 plate and comprehensive observation provides a practical strategy for increasing reliability of single-cell origin of clonal cells. Cellular heterogeneity seems to be an intrinsic feature of dental pulp cells.

Modern translational medicine, pharmacology and other fields require effective skin cryopreservation technology. Vitrification is promising technology for this aim. This rapid freezing technique minimizes hexagonal ice crystals formation, time of stress condition for cells in samples and dehydration both in the cytoplasm of cells and in the extracellular space. This study is aimed at finding the optimal combination of extracellular (sucrose, mannitol, polyethylene glycol and FBS) and intracellular (DMSO, glycerol, ethylene glycol) cryoprotective agents to achieve the highest viability and tissue structure preservation of skin samples after vitrification.

Cellular protrusions mediated by the membrane-deforming I-BAR domain protein IRSp53 are involved in cell migration, including metastasis. However, the role of IRSp53 in cell proliferation remains unclear. Here, we examined the role of IRSp53 in cell proliferation and found that it acts through secretion. Coculture of gingiva squamous carcinoma Ca9-22 cells and their IRSp53-knockout cells restored proliferation to parental Ca9-22 cell levels, suggesting possible secretion dependent on IRSp53. Notably, the amounts of microvesicle fraction proteins that were secreted into the culture medium were reduced in the IRSp53-knockout cells. The IRSp53-knockout cells exhibited decreased phosphorylation of mitogen-activated protein kinase, suggesting the decrease in the proliferation signals. The phosphorylation was restored by the addition of the microvesicles. In mice xenograft Ca9-22 cells, IRSp53-containing particles were secreted around the xenograft, indicating that IRSp53-dependent secretion occurs in vivo. In a tumor mice model, IRSp53 deficiency elongated lifespan. In some human cancers, the higher levels of IRSp53 mRNA expression was found to be correlated with shorter survival years. Therefore, IRSp53 is involved in tumor progression and secretion for cellular proliferation.

Endosomal sorting complex required for transport (ESCRT-III) is a membrane remodeling complex involved in a large number of cellular processes. It appears to perform an essential function in eukaryotes, since to date no eukaryotic organism completely devoid of ESCRT-III has been found. Yet, yeast cells with a deletion of all eight known ESCRT-III genes are viable. We therefore searched for new, previously undiscovered ESCRT-III like proteins in yeast. HHPred uncovered several proteins with similarity to Snf7. The similarity was mostly restricted to the 1-2 hairpin region of Snf7. A conserved pattern of amino acids was detected in this region. The protein encoded by ORF YPL199c strikingly resembled Snf7 in its secondary structure. Since this protein could be the ninth member of the ESCRT-III family in yeast, we called it Nbr9 ("number nine"). Nbr9 is palmitoylated and localizes to the plasma membrane. In contrast to other palmitoylated proteins, it is not associated with lipid rafts. When NBR9 was deleted in the octuple ESCRT-III deletion background, the yeast cells were still viable. However, despite a number of experiments, we do not have evidence at present that Nbr9 is part of an alternative ESCRT-III complex.

Bazooka/Par-3 (Baz) is an evolutionarily conserved scaffold protein that together with its binding partners atypical protein kinase C (aPKC) and Par-6 forms the Par complex. This protein complex functions as a master regulator for the establishment and maintenance of cell polarity in many different cell types. In the vast majority of published research papers Par complex members have been reported to localize at the cell cortex and at intercellular junctions. However, there have also been several reports claiming localization and function of Par complex members at additional subcellular sites, in particular the nucleus, the nuclear envelope and the neuromuscular junction. In this study we have re-assessed this issue for Baz in a systematic manner. We used a variety of antibodies raised in different host animals against different epitopes of Baz for confocal imaging of Drosophila tissues. We tested the specificity of these antisera by using mosaic analysis with null mutant baz alleles and tissue-specific RNAi against baz. In addition, we used a GFP-tagged gene trap line for Baz and a bacterial artificial chromosome (BAC) expressing GFP-tagged Baz under control of its endogenous promoter in a baz null mutant background to compare the subcellular localization of the respective GFP-Baz fusion proteins to the staining results with anti-Baz antisera. Together, these experiments did not provide any evidence for specific localization of Baz to the nucleus or the neuromuscular junction.

Optimization of skin cryopreservation technology is an important task for translational medicine, pharmacology, and a list of other fields. One of the promising approaches is vitrification. This rapid freezing technique allows one to ensure minimal damage to the sample by equating the formation of ice crystals and dehydration both in the cytoplasm of cells and in the extracellular space. This study is aimed at finding the optimal combination of cryoprotective agents (DMSO, glycerol, sucrose and polyethylene glycol) to achieve the highest viability of skin samples after vitrification.

Investigations of structural organization of DNA for stressed (increased stress resistance), anabiotic and mummified cells obtained by introducing 4-hexylresorcinol at different concentrations for different stages of cell culture growth using diffraction of synchrotron radiation were done. Experimental studies allow us to conclude that 4-hexylresorcinol is the initiator of the cell transition to the anabiotic and mummified states at stationary state. At pre-stationary state of growth 4-hexylresorcinol initiates a transition of cells to a mummified state but not to anabiotic one. The structure of DNA within a cell in the anabiotic dormant state (practically complete absence of metabolism) and the dormant state (starvation stress) coincide (forms nanocrystalline structures). Our data suggest the universality of DNA condensation or the universality of DNA protection by a protein Dps for dormant state, regardless of the type of stress. Mummified state (the complete absence of metabolism) is very different in structure (has no ordering within a cell).

BackgroundThe prevalence of chronic rhinosinusitis (CRS) has been reported to range from 5% to 12% in different parts of the world. However, the prevalence of CRS in Thailand has not been investigated. Our objective was to determine the prevalence of CRS among hospital workers and identify any potential problems encountered during the survey. MethodAdult workers (>18 years) of Rajavithi Hospital, a tertiary hospital in Bangkok, Thailand, were recruited. This cross-sectional survey was conducted from September 2021 to September 2022. The participants were given a link to online questionnaires asking if they had nasal obstruction, discharge, decreased smell sensation, or facial pain and their respective duration. Phone numbers and email were asked for contact when needed. The participants who fulfilled the symptom and duration according to the European position paper on rhinosinusitis and nasal polyp 2020 were counted as symptom-based CRS patients. These patients were then contacted for nasal endoscopy. The endoscopic-based diagnosis of CRS was made after positive endoscopy findings. ResultA total of 1,025 participants (mean age of 33.7 years) were recruited. Of total, there were 34 (3.3%) and 6 (0.58%) participants fulfilled symptom- and endoscopy-based diagnoses, respectively. Fourteen participants did not respond to calls or emails. Five patients refused to visit the clinic due to inconveniences. One patient refused nasal endoscopy because of the expenses. ConclusionThe overall prevalence of chronic rhinosinusitis in workers of Rajavithi Hospital, was 3.3% and 0.58% by symptom- and endoscopy-based criteria, respectively. Difficulty reaching the participants for nasal endoscopy was the leading survey problem.

Polarity is one of the fundamental properties of cells. It is characterised by the asymmetric distribution of lipids and proteins in the cell membrane and cortex, and organelles in the cell. Cell polarisation drives essential processes such as morphogenesis, cell migration, asymmetric cell division, directional transport of molecules across epithelium, nerve impulse transmission etc. Although, the importance of polarity proteins such as atypical protein kinase C (aPKC) and Lgl2 (lethal giant larvae 2) has been relatively well understood in the developing zebrafish epidermis, whether basal lamina components are important for polarity maintenance has remained unclear. I analysed the role of Laminin5 (basal lamina component) in the establishment and maintenance of apicobasal polarity, in developing bi layered zebrafish epidermis. I found that the loss of laminin 5 function results in reduced E cadherin localization and increased cell spreading along with dynamic cell boundaries and increased cell proliferation indicating acquisition of mesenchymal traits. A similar phenotype was observed in integrin 6b mutant, which did not exacerbate in the double mutant embryos indicating that laminin 5 and integrin 6b function in the same pathway. Interestingly, periderm, the cell layer above basal epidermis, maintains its apicobasal polarity and epithelial integrity presumably via reinforcing the localization of aPKC and Lgl. My work unravels the importance of Laminin 5 and Integrin 6b interaction in the maintenance of epithelial characteristics in the basal layer of the developing zebrafish epidermis.

In recent years, the interaction between cells and the extracellular matrix (ECM) has become a new focus in understanding tissue morphogenesis, regeneration, and disease. However, the lack of specific techniques to study the ECM composition in preserved tissue structures remains a major obstacle to explaining ECM changes in response to extrinsic stimuli. To overcome this, we propose a novel strategy that uses multidimensional fluorescence microscopy and computational tools to quantify ECM composition in immunolabeled tissues and/or cell-derived matrices (CDM). This approach includes a detailed protocol for densitometric fluorescence calibration and procedures for image acquisition, processing, and automated quantification. Using this method, we present new data comparing collagen types I, III, and IV, and fibronectin contents in various tissues. These results emphasize the importance of studying ECM composition in situ under both normal homeostatic and disease conditions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/535641v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1203e08org.highwire.dtl.DTLVardef@1c88c1borg.highwire.dtl.DTLVardef@1664ac2org.highwire.dtl.DTLVardef@b63154_HPS_FORMAT_FIGEXP M_FIG C_FIG

Myosin-X (MYO10) is an actin-based motor protein involved in cytoskeletal dynamics, membrane interactions, and integrin-mediated adhesion. To investigate MYO10s cellular roles, we generated MYO10 knockdown (MYO10KD) HeLa and COS7 cell lines using lentiviral shRNA. Compared to wild-type cells, both MYO10KD lines showed reduced proliferation and impaired cell migration in wound assays. There were fewer edge filopodia in HeLa cells. Additionally, MYO10KD cells demonstrated increased spreading on laminin-coated substrates, suggesting altered integrin activation and cytoskeletal linkage. Our results reinforce MYO10s importance in cell proliferation, adhesion, and migration; these MYO10KD lines provide an accessible cell culture model for further study of MYO10.