Motile cilia are found in numerous locations throughout our body and play a critical role in various physiological processes. The most commonly used method to assess cilia motility is to quantify ...cilia beat frequency (CBF) via video microscopy. However, a large heterogeneity exists within published literature regarding the framerate used to image cilia motility for calculating CBF. The aim of this study was to determine the optimal frame rate required to image cilia motility for CBF assessment, and if the Nyquist theorem may be used to set this rate. One‐second movies of cilia were collected at >600 fps from mouse airways and ependyma at room‐temperature or 37°C. Movies were then down‐sampled to 30–300 fps. CBF was quantified for identical cilia at different framerates by either manual counting or automated MATLAB script. Airway CBF was significantly impaired in 30 fps movies, while ependymal CBF was significantly impaired in both 60 and 30 fps movies. Pairwise comparison showed that video framerate should be at least 150 fps to accurately measure CBF, with minimal improvement in CBF accuracy in movies >150 fps. The automated script was also found to be less accurate for measuring CBF in lower fps movies than manual counting, however, this difference disappeared in higher framerate movies (>150 fps). In conclusion, our data suggest the Nyquist theorem is unreliable for setting sampling rate for CBF measurement. Instead, sampling rate should be 3–4 times faster than CBF for accurate CBF assessment. Especially if CBF calculation is to be automated.
The aim of this study was to determine the optimal frame rate required to image cilia motility for cilia beat frequency assessment, and if the Nyquist theorem may be used to set this rate. our data suggests the Nyquist theorem is unreliable for setting imaging sampling rate for cilia beat frequency measurement. Instead, sampling rate should be 3–4 times faster than cilia beat frequency for accurate CBF assessment.
Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. ...Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than their absolute activation state, highlighting an unappreciated interplay between these modifications. Mechanistic analysis of the redox regulation of Akt identified two cysteine residues in the pleckstrin homology domain (C60 and C77) to be reversibly oxidized. Oxidation at these sites affected Akt recruitment to the plasma membrane by stabilizing the PIP
binding pocket. Our data provide insights into the interplay between oxidative stress-derived redox signalling and protein phosphorylation networks and serve as a resource for understanding the contribution of cellular oxidation to a range of diseases.
Adipose tissue is essential for whole-body glucose homeostasis, with a primary role in lipid storage. It has been previously observed that lactate production is also an important metabolic feature of ...adipocytes, but its relationship to adipose and whole-body glucose disposal remains unclear. Therefore, using a combination of metabolic labeling techniques, here we closely examined lactate production of cultured and primary mammalian adipocytes. Insulin treatment increased glucose uptake and conversion to lactate, with the latter responding more to insulin than did other metabolic fates of glucose. However, lactate production did not just serve as a mechanism to dispose of excess glucose, because we also observed that lactate production in adipocytes did not solely depend on glucose availability and even occurred independently of glucose metabolism. This suggests that lactate production is prioritized in adipocytes. Furthermore, knocking down lactate dehydrogenase specifically in the fat body of Drosophila flies lowered circulating lactate and improved whole-body glucose disposal. These results emphasize that lactate production is an additional metabolic role of adipose tissue beyond lipid storage and release.
Many cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable ...communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases, suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development, but our molecular understanding of the precise glycans, catalytic enzymes, and lectins involved remains only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown digalactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labeling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins, most notably the upregulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation, suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the upregulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.
Display omitted
•Proteomic, glycomic and glycoproteomic analysis of myogenesis.•Mechanistic insights into site-specific glycoproteome regulation via discrete glycosidases and glycosyltransferases.•Quantification and validation of glycan-binding proteins.•Functional analysis of LGALS1 reveals a role in myogenesis and muscle development.
The proteome, N-glycome, and N-glycoproteome have been determined during myotube formation. This revealed a complex regulation of N-glycosylation including a switch in sialylation linkages, digalactosylation, and paucimannosylation on receptors and adhesion molecules. The data also revealed regulation of several glycan-binding proteins including LGALS1, and functional validation revealed a role of this protein in myogenesis and skeletal muscle development.
Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential ...roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Metabolism is often studied in animal models, with the
fruit fly model offering ease of genetic manipulation and high-throughput studies. Fly metabolism is typically studied using end-point assays ...that are simple but destructive, and do not provide information on the utilization of specific nutrients. To address these limitations, we adapted existing gas-trapping protocols to measure the oxidation of radiolabeled substrates (such as glucose) in multi-well plates. This protocol is cost effective, simple, and offers precise control over experimental diet and measurement time, thus being amenable to high-throughput studies. Furthermore, it is nondestructive, enabling time-course experiments and multiplexing with other parameters. Overall, this protocol is useful for merging fly genetics with metabolic studies to understand whole organism responses to different macronutrients.
Meckel-Gruber syndrome (MKS) is a recessive disorder resulting in multiple birth defects that are associated with mutations affecting ciliogenesis. We recovered a mouse mutant with a mutation in the ...Mks1 gene (Mks1(del64-323)) that caused a 260-amino-acid deletion spanning nine amino acids in the B9 domain, a protein motif with unknown function conserved in two other basal body proteins. We showed that, in wild-type cells, Mks1 was localized to the mother centriole from which the cilium was generated. However, in mutant Mks1(del64-323) cells, Mks1 was not localized to the centriole, even though it maintained a punctate distribution. Resembling MKS patients, Mks1 mutants had craniofacial defects, polydactyly, congenital heart defects, polycystic kidneys and randomized left-right patterning. These defects reflected disturbance of functions subserved by motile and non-motile cilia. In the kidney, glomerular and tubule cysts were observed along with short cilia, and cilia were reduced in number to a near-complete loss. Underlying the left-right patterning defects were fewer and shorter nodal cilia, and analysis with fluorescent beads showed no directional flow at the embryonic node. In the cochlea, the stereocilia were mal-patterned, with the kinocilia being abnormally positioned. Together, these defects suggested disruption of planar cell polarity, which is known to regulate node, kidney and cochlea development. In addition, we also showed that Shh signaling was disrupted. Thus, in the neural tube, the floor plate was not specified posteriorly even as expression of the Shh mediator Gli2 increased. By contrast, the Shh signaling domain was expanded in the anterior neural tube and anterior limb bud, consistent with reduced Gli3-repressor (Gli3R) function. The latter probably accounted for the preaxial digit duplication exhibited by the Mks1(del64-323) mutants. Overall, these findings indicate that centriole localization of Mks1 is required for ciliogenesis of motile and non-motile cilia, but not for centriole assembly. On the basis of these results, we hypothesize a role for the B9 domain in mother centriole targeting, a possibility that warrants further future investigations.
Abstract
Genetic and environmental factors play a major role in metabolic health. However, they do not act in isolation, as a change in an environmental factor such as diet may exert different ...effects based on an individual’s genotype. Here, we sought to understand how such gene–diet interactions influenced nutrient storage and utilization, a major determinant of metabolic disease. We subjected 178 inbred strains from the Drosophila genetic reference panel (DGRP) to diets varying in sugar, fat, and protein. We assessed starvation resistance, a holistic phenotype of nutrient storage and utilization that can be robustly measured. Diet influenced the starvation resistance of most strains, but the effect varied markedly between strains such that some displayed better survival on a high carbohydrate diet (HCD) compared to a high-fat diet while others had opposing responses, illustrating a considerable gene × diet interaction. This demonstrates that genetics plays a major role in diet responses. Furthermore, heritability analysis revealed that the greatest genetic variability arose from diets either high in sugar or high in protein. To uncover the genetic variants that contribute to the heterogeneity in starvation resistance, we mapped 566 diet-responsive SNPs in 293 genes, 174 of which have human orthologs. Using whole-body knockdown, we identified two genes that were required for glucose tolerance, storage, and utilization. Strikingly, flies in which the expression of one of these genes, CG4607 a putative homolog of a mammalian glucose transporter, was reduced at the whole-body level, displayed lethality on a HCD. This study provides evidence that there is a strong interplay between diet and genetics in governing survival in response to starvation, a surrogate measure of nutrient storage efficiency and obesity. It is likely that a similar principle applies to higher organisms thus supporting the case for nutrigenomics as an important health strategy.
The terminal cells of the larval Drosophila tracheal system extend dozens of branched cellular processes, most of which become hollow intracellular tubes that support gas exchange with internal ...tissues. Previously, we undertook a forward genetic mosaic screen to uncover the pathways regulating terminal cell size, morphogenesis, and the generation and maintenance of new intracellular tubes. Our initial work identified several mutations affecting terminal cell size and branch number, and suggested that branch complexity and cell size are typically coupled but could be genetically separated. To deepen our understanding of these processes, we have further characterized and determined the molecular identities of mutations in the genes sprout, denuded and asthmatic, that had been implicated in our initial screen. Here we reveal the molecular identity of these genes and describe their function in the context of the TOR and Hippo pathways, which are widely appreciated to be key regulators of cell and organ size.
•Terminal cell size and branching require E2F1-dependent endoreplication.•The terminal cell size mutant sprout is an allele if InR.•The terminal cell size and branch number mutant denuded is an allele of ATAD3A.•Mutations in zif also affect terminal cell size and branch number.
Larval terminal cells of the Drosophila tracheal system generate extensive branched tubes, requiring a huge increase in apical membrane. We discovered that terminal cells compromised for apical ...membrane expansion - mTOR-vATPase axis and apical polarity mutants - were invaded by the neighboring stalk cell. The invading cell grows and branches, replacing the original single intercellular junction between stalk and terminal cell with multiple intercellular junctions. Here, we characterize disjointed, a mutation in the same phenotypic class. We find that disjointed encodes Drosophila Archease, which is required for the RNA ligase (RtcB) function that is essential for tRNA maturation and for endoplasmic reticulum stress-regulated nonconventional splicing of Xbp1 mRNA. We show that the steady-state subcellular localization of Archease is principally nuclear and dependent upon TOR-vATPase activity. In tracheal cells mutant for Rheb or vATPase loci, Archease localization shifted dramatically from nucleus to cytoplasm. Further, we found that blocking tRNA maturation by knockdown of tRNAseZ also induced compensatory branching. Taken together, these data suggest that the TOR-vATPase axis promotes apical membrane growth in part through nuclear localization of Archease, where Archease is required for tRNA maturation.
PDF
