Acto3D: an open-source user-friendly volume rendering software for high-resolution 3D fluorescence imaging in biology.

Advances in fluorescence microscopy and tissue-clearing have revolutionised 3D imaging of fluorescently labelled tissues, organs and embryos. However, the complexity and high cost of existing software and computing solutions limit their widespread adoption, especially by researchers with limited resources. Here, we present Acto3D, an open-source software, designed to streamline the generation and analysis of high-resolution 3D images of targets labelled with multiple fluorescent probes. Acto3D provides an intuitive interface for easy 3D data import and visualisation. Although Acto3D offers straightforward 3D viewing, it performs all computations explicitly, giving users detailed control over the displayed images. Leveraging an integrated graphics processing unit, Acto3D deploys all pixel data to system memory, reducing visualisation latency. This approach facilitates accurate image reconstruction and efficient data processing in 3D, eliminating the need for expensive high-performance computers and dedicated graphics processing units. We have also introduced a method for efficiently extracting lumen structures in 3D. We have validated Acto3D by imaging mouse embryonic structures and by performing 3D reconstruction of pharyngeal arch arteries while preserving fluorescence information. Acto3D is a cost-effective and efficient platform for biological research.

Tubular cell loss in early inv/nphp2 mutant kidneys represents a possible homeostatic mechanism in cortical tubular formation.

Inversion of embryonic turning (inv) cystic mice develop multiple renal cysts and are a model for type II nephronophthisis (NPHP2). The defect of planar cell polarity (PCP) by oriented cell division was proposed as the underlying cellular phenotype, while abnormal cell proliferation and apoptosis occur in some polycystic kidney disease models. However, how these cystogenic phenotypes are linked and what is most critical for cystogenesis remain largely unknown. In particular, in early cortical cytogenesis in the inv mutant cystic model, it remains uncertain whether the increased proliferation index results from changes in cell cycle length or cell fate determination. To address tubular cell kinetics, doubling time and total number of tubular cells, as well as amount of genomic DNA (gDNA), were measured in mutant and normal control kidneys. Despite a significantly higher bromodeoxyuridine (BrdU)-proliferation index in the mutant, total tubular cell number and doubling time were unaffected. Unexpectedly, the mutant had tubular cell loss, characterized by a temporal decrease in tubular cells incorporating 5-ethynyl-2´-deoxyuridine (EdU) and significantly increased nuclear debris. Based on current data we established a new multi-population shift model in postnatal renal development, indicating that a few restricted tubular cell populations contribute to cortical tubular formation. As in the inv mutant phenotype, the model simulation revealed a large population of tubular cells with rapid cell cycling and tubular cell loss. The proposed cellular kinetics suggest not only the underlying mechanism of the inv mutant phenotype but also a possible renal homeostatic mechanism for tubule formation.

Maintenance of pluripotency in mouse ES cells without Trp53.

Tumor suppressor Trp53 works as a guardian of the genome in somatic cells. In mouse embryonic stem (ES) cells, it was reported that Trp53 represses pluripotency-associated transcription factor Nanog to induce differentiation. However, since Trp53-null mice develop to term, Trp53 is dispensable for both the maintenance and differentiation of the pluripotent stem cell population in vivo, suggesting the differential functions of Trp53 in ES cells and embryos. To reveal the basis of this discrepancy, here we established a new line of Trp53-null ES cells by sequential gene targeting and evaluated their ability to differentiate in vitro and in vivo. We found that Trp53-null ES cells had defects in differentiation in vitro as reported previously, whereas they were able to contribute to normal development in chimeric embryos. These data indicated that the requirement of Trp53 for maintaining and executing the ES pluripotency is not absolute.

DNA methylation restricts lineage-specific functions of transcription factor Gata4 during embryonic stem cell differentiation.

DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES) cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Gata4-binding-site profiles and DNA methylation analyses suggested that DNA methylation modulates the Gata4 response through diverse mechanisms. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells.

Functionally defined substates within the human embryonic stem cell compartment.

Human embryonic stem (ES) cells can undergo spontaneously differentiation in standard culture conditions, demonstrating that the undifferentiated state is relatively unstable. The heterogeneous expression of SSEA3 observed within human ES colonies, provides a means to examine undifferentiated stem cell substates. Through functional testing of single cells we have shown that undifferentiated ES cells can be segregated into functionally discrete subpopulations on the basis of SSEA3 expression: SSEA3(High), SSEA(Low) and SSEA3(Negative). Human ES subpopulations were found to be interconvertible, but they possess distinct properties when challenged to differentiate along the neural lineage. These data suggest that ES cells with pluripotent/self-renewal capacities can exhibit different responses to induction of differentiation.

Carbohydrate binding specificity of a fucose-specific lectin from Aspergillus oryzae: a novel probe for core fucose.

The alpha1,6-fucosyl residue (core fucose) of glycoproteins is widely distributed in mammalian tissues and is altered under pathological conditions. A probe that specifically detects core fucose is important for understanding the role of this oligosaccharide structure. Aleuria aurantia lectin (AAL) and Lens culimaris agglutinin-A (LCA) have been often used as carbohydrate probes for core fucose in glycoproteins. Here we show, by using surface plasmon resonance (SPR) analysis, that Aspergillus oryzae l-fucose-specific lectin (AOL) has strongest preference for the alpha1,6-fucosylated chain among alpha1,2-, alpha1,3-, alpha1,4-, and alpha1,6-fucosylated pyridylaminated (PA)-sugar chains. These results suggest that AOL is a novel probe for detecting core fucose in glycoproteins on the surface of animal cells. A comparison of the carbohydrate-binding specificity of AOL, AAL, and LCA by SPR showed that the irreversible binding of AOL to the alpha1,2-fucosylated PA-sugar chain (H antigen) relative to the alpha1,6-fucosylated chain was weaker than that of AAL, and that the interactions of AOL and AAL with alpha1,6-fucosylated glycopeptide (FGP), which is considered more similar to in vivo glycoproteins than PA-sugar chains, were similar to their interactions with the alpha1,6-fucosylated PA-sugar chain. Furthermore, positive staining of AOL, but not AAL, was completely abolished in the cultured embryo fibroblast (MEF) cells obtained from alpha1,6-fucosyltransferase (Fut8) knock-out mice, as assessed by cytological staining. Taken together, these results suggest that AOL is more suitable for detecting core fucose than AAL or LCA.

Man2C1, an alpha-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol.

The endoplasmic-reticulum-associated degradation of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the endoplasmic reticulum and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, they are deglycosylated by the PNGase (peptide:N-glycanase). The free oligosaccharides released by PNGase are known to be further catabolized by a cytosolic alpha-mannosidase, although the gene encoding this enzyme has not been identified unequivocally. The findings in the present study demonstrate that an alpha-mannosidase, Man2C1, is involved in the processing of free oligosaccharides that are formed in the cytosol. When the human Man2C1 orthologue was expressed in HEK-293 cells, most of the enzyme was localized in the cytosol. Its activity was enhanced by Co2+, typical of other known cytosolic alpha-mannosidases so far characterized from animal cells. The down-regulation of Man2C1 activity by a small interfering RNA drastically changed the amount and structure of oligosaccharides accumulating in the cytosol, demonstrating that Man2C1 indeed is involved in free oligosaccharide processing in the cytosol. The oligosaccharide processing in the cytosol by PNGase, endo-beta-N-acetylglucosaminidase and alpha-mannosidase may represent the common 'non-lysosomal' catabolic pathway for N-glycans in animal cells, although the molecular mechanism as well as the functional importance of such processes remains to be determined.

beta1,4-N-Acetylglucosaminyltransferase III potentiates beta1 integrin-mediated neuritogenesis induced by serum deprivation in Neuro2a cells.

Aspects of the biological significance of the bisecting N-acetylglucosamine (GlcNAc) structure on N-glycans introduced by beta1,4-N-acetylglucosaminyltransferase III (GnT-III) in Neuro2a cell differentiation are demonstrated. The overexpression of GnT-III in the cells led to the induction of axon-like processes with numerous neurites and swellings, in which beta1 integrin was localized, under conditions of serum starvation. This enhancement in neuritogenesis was suppressed by either the addition of a bisecting GlcNAc-containing N-glycan or erythroagglutinating phytohemagglutinin (E(4)-PHA), which preferentially recognizes the bisecting GlcNAc. GnT-III-promoted neuritogenesis was also significantly perturbed by treatment with a functional blocking anti-beta1 integrin antibody. In fact, beta1 integrin was found to be one of the target proteins of GnT-III, as confirmed by a pull-down assay with E(4)-PHA. These data suggest that N-glycans with a bisecting GlcNAc on target molecules, such as beta1 integrin, play important roles in the regulation of neuritogenesis.

Cellular stresses induce the nuclear accumulation of importin alpha and cause a conventional nuclear import block.

We report here that importin alpha accumulates reversibly in the nucleus in response to cellular stresses including UV irradiation, oxidative stress, and heat shock. The nuclear accumulation of importin alpha appears to be triggered by a collapse in the Ran gradient, resulting in the suppression of the nuclear export of importin alpha. In addition, nuclear retention and the importin beta/Ran-independent import of importin alpha also facilitate its rapid nuclear accumulation. The findings herein show that the classical nuclear import pathway is down-regulated via the removal of importin alpha from the cytoplasm in response to stress. Moreover, whereas the nuclear accumulation of heat shock cognate 70 is more sensitive to heat shock than the other stresses, importin alpha is able to accumulate in the nucleus at all the stress conditions tested. These findings suggest that the stress-induced nuclear accumulation of importin alpha can be involved in a common physiological response to various stress conditions.

CD151 regulates epithelial cell-cell adhesion through PKC- and Cdc42-dependent actin cytoskeletal reorganization.

CD151, a member of the tetraspanin family proteins, tightly associates with integrin alpha3beta1 and localizes at basolateral surfaces of epithelial cells. We found that overexpression of CD151 in A431 cells accelerated intercellular adhesion, whereas treatment of cells with anti-CD151 mAb perturbed the integrity of cortical actin filaments and cell polarity. E-Cadherin puncta formation, indicative of filopodia-based adhesion zipper formation, as well as E-cadherin anchorage to detergent-insoluble cytoskeletal matrix, was enhanced in CD151-overexpressing cells. Levels of GTP-bound Cdc42 and Rac were also elevated in CD151-overexpressing cells, suggesting the role of CD151 in E-cadherin-mediated cell-cell adhesion as a modulator of actin cytoskeletal reorganization. Consistent with this possibility, engagement of CD151 by the substrate-adsorbed anti-CD151 mAb induced prominent Cdc42-dependent filopodial extension, which along with E-cadherin puncta formation, was strongly inhibited by calphostin C, a protein kinase C (PKC) inhibitor. Together, these results indicate that CD151 is involved in epithelial cell-cell adhesion as a modulator of PKC- and Cdc42-dependent actin cytoskeletal reorganization.

Expiratory asynchrony in proportional assist ventilation.

One of the proposed advantages of proportional assist ventilation (PAV) has been the automatic synchrony between the end of the patient's inspiratory effort and the ventilator cycle (i.e., expiratory synchrony). However, recent clinical studies have shown a prolonged ventilator inspiratory time or even a "runaway" phenomenon with the normal use of PAV. We hypothesize that control-system delay may account for this, because in reality there is always some degree of delays between control-system's input and output in all ventilators. Computer simulation study to date has not taken into account the potential effect of control-system delay on expiratory synchrony. We therefore created a computer model in which the parameter of control-system delay time was introduced. We found that significant expiratory asynchrony may occur with this more realistic model of PAV. The ventilator flow termination may fall behind the completion of the patient inspiration by as long as 0.33 seconds under the selected simulation conditions. The inspiratory termination delay time is in proportion to the control-system delay time, the respiratory time constant, and the assist gain settings. In conclusion, this model indicates that due to the unavoidable control-system delay in the ventilators, expiratory asynchrony may be an inherent shortcoming associated with PAV.