Hemodynamic shear stress from blood flow on the endothelium critically regulates vascular function in many physiological and pathological situations. Endothelial cells adapt to shear stress by remodeling their cytoskeletal components and subsequently by changing their shape and orientation. We demonstrate that β1 integrin activation is critically controlled during the mechanoresponse of endothelial cells to shear stress. Indeed, we show that overexpression of the CCM complex, an inhibitor of β1 integrin activation, blocks endothelial actin rearrangement and cell reorientation in response to shear stress similarly to β1 integrin silencing. Conversely, depletion of CCM2 protein leads to an elongated “shear-stress-like” phenotype even in the absence of flow. Taken together, our findings reveal the existence of a balance between positive extracellular and negative intracellular signals, i.e. shear stress and CCM complex, for the control of β1 integrin activation and subsequent adaptation of vascular endothelial cells to mechanostimulation by fluid shear stress.
β1 integrin; CCM; Shear stress; Mechanotransduction
Patients suffering from glycogen storage disease V (McArdle disease) were shown to have higher surface electrical activity in their skeletal muscles when exercising at the same intensity as their healthy counterparts, indicating more muscle fibre recruitment. To explain this phenomenon, this study investigated whether muscle fibre type is shifted towards a predominance in type I fibres as a consequence of the disease. Muscle biopsies from the Biceps brachii (BB) (n = 9) or Vastus lateralis (VL) (n = 8) were collected over a 13-year period from male and female patients diagnosed with McArdle disease, analysed for myosin heavy chain (MHC) isoform content using SDS-PAGE, and compared to healthy controls (BB: n = 3; VL: n = 10). All three isoforms were expressed and no difference in isoform expression in VL was found between the McArdle patients and healthy controls (MHC I: 33±19% vs. 43±7%; MHC IIa: 52±9% vs. 40±7%; MHC IIx: 15±18% vs. 17±9%). Similarly, the BB isoform content was also not different between the two groups (MHC I: 33±14% vs. 30±11%; MHC IIa: 46±17% vs. 39±5%; MHC IIx: 21±13% vs. 31±14%). In conclusion, fibre type distribution does not seem to explain the higher surface EMG in McArdle patients. Future studies need to investigate muscle fibre size and contractility of McArdle patients.
Myosin heavy chain; Glycogen storage disease V; Phosphorylase deficiency
Adult stem cells face the challenge of maintaining tissue homeostasis by self-renewal while maintaining their proliferation potential over the lifetime of an organism. Continuous proliferation can cause genotoxic/metabolic stress that can compromise the genomic integrity of stem cells. To prevent stem cell exhaustion, highly proliferative adult tissues maintain a pool of quiescent stem cells that divide only in response to injury and thus remain protected from genotoxic stress. Hydra is a remarkable organism with highly proliferative stem cells and ability to regenerate at whole animal level. Intriguingly, hydra does not display consequences of high proliferation, such as senescence or tumour formation. In this study, we investigate if hydra harbours a pool of slow-cycling stem cells that could help prevent undesirable consequences of continuous proliferation. Hydra were pulsed with the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU) and then chased in the absence of EdU to monitor the presence of EdU-retaining cells. A significant number of undifferentiated cells of all three lineages in hydra retained EdU for about 8–10 cell cycles, indicating that these cells did not enter cell cycle. These label-retaining cells were resistant to hydroxyurea treatment and were predominantly in the G2 phase of cell cycle. Most significantly, similar to mammalian quiescent stem cells, these cells rapidly entered cell division during head regeneration. This study shows for the first time that, contrary to current beliefs, cells in hydra display heterogeneity in their cell cycle potential and the slow-cycling cells in this population enter cell cycle during head regeneration. These results suggest an early evolution of slow-cycling stem cells in multicellular animals.
Hydra; Quiescence; Regeneration; Stem cells; Cnidaria
Mate choice is an important ecological behavior in fish, and is often based on visual cues of body patterns. The Crown Butterfly fish Chaetodon paucifasciatus (Chaetodontidae) is a monogamist, territorial species; it swims in close proximity to its partner throughout most of its life. This species is characterized by a pattern of 6–8 vertical black stripes on a white background, on both sides of its body. Our aim was to define spatial features (variations) in body patterns by evaluating the level of dissimilarity between both sides of each individual fish, and the level of dissimilarity between patterns of different individuals. In addition, we tested whether the fish are attracted to or reject specific features of the body patterns. Features were defined and counted using photographs of body patterns. Attraction to or rejection of specific features were tested behaviorally using a dual-choice experiment of video animations of individuals swimming over a coral-reef background. We found that the patterns of each fish and sides of the body were no less dissimilar, compared intraspecificly to other fish, and that each side pattern was unique and distinguishable. Variations in the patterns occurred mostly in the last three posterior stripes. Individuals were mainly attracted to conspecifics with multiple crossing patterns (two or more consecutive crossings), and rejected patterns with holes. Our results suggest that in this species the unique body pattern of each fish is used for conspecific identification of mates and intruders.
Animal recognition; Vision; Sexual selection; Computer animation; Symmetry
Adducin is a ubiquitously expressed actin- and spectrin-binding protein involved in cytoskeleton organization, and is regulated through phosphorylation of the myristoylated alanine-rich C-terminal kinase (MARCKS)-homology domain by protein kinase C (PKC). We have previously shown that the Drosophila adducin, Hu-li tai shao (Hts), plays a role in larval neuromuscular junction (NMJ) growth. Here, we find that the predominant isoforms of Hts at the NMJ contain the MARCKS-homology domain, which is important for interactions with Discs large (Dlg) and phosphatidylinositol 4,5-bisphosphate (PIP2). Through the use of Proximity Ligation Assay (PLA), we show that the adducin-like Hts isoforms are in complexes with Dlg and PIP2 at the NMJ. We provide evidence that Hts promotes the phosphorylation and delocalization of Dlg at the NMJ through regulation of the transcript distribution of the PAR-1 and CaMKII kinases in the muscle. We also show that Hts interactions with Dlg and PIP2 are impeded through phosphorylation of the MARCKS-homology domain. These results are further evidence that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila.
Dlg; Drosophila; Hts; PIP2; Adducin; Neuromuscular junction
NANOS2 and NANOS3 belong to the Nanos family of proteins that contain a conserved zinc finger domain, which consists of two consecutive CCHC-type zinc finger motifs, and contribute to germ cell development in mice. Previous studies indicate that there are redundant and distinct functions of these two proteins. NANOS2 rescues NANOS3 functions in the maintenance of primordial germ cells, whereas NANOS3 fails to replace NANOS2 functions in the male germ cell pathway. However, the lack of a conditional allele of Nanos3 has hampered delineation of each contribution of NANOS2 and NANOS3 to the male germ cell pathway. In addition, the molecular mechanism underlying the distinct functions of these proteins remains unexplored. Here, we report an unexpected observation of a transgenic mouse line expressing a NANOS2 variant harboring mutations in the zinc finger domain. Transcription of Nanos2 and Nanos3 was strongly compromised in the presence of this transgene, which resulted in the mimicking of the Nanos2/Nanos3 double-null condition in the male gonad. In these transgenic mice, P-bodies involved in RNA metabolism had disappeared and germ cell differentiation was more severely affected than that in Nanos2-null mice, indicating that NANOS3 partially substitutes for NANOS2 functions. In addition, similar to NANOS2, we found that NANOS3 associated with the CCR4-NOT deadenylation complex but via a direct interaction with CNOT8, unlike CNOT1 in the case of NANOS2. This alternate interaction might account for the molecular basis of the functional redundancy and differences in NANOS2 and NANOS3 functions.
Nanos; CCR4-NOT deadenylase; Germ cell
Spindle length varies dramatically across species and during early development to segregate chromosomes optimally. Both intrinsic factors, such as regulatory molecules, and extrinsic factors, such as cytoplasmic volume, determine spindle length scaling. However, the properties that govern spindle shape and whether these features can be modulated remain unknown. Here, we analyzed quantitatively how the molecular players which regulate microtubule dynamics control the kinetics of spindle formation and shape. We find that, in absence of Clasp1 and Clasp2, spindle assembly is biphasic due to unopposed inward pulling forces from the kinetochore-fibers and that kinetochore-fibers also alter spindle geometry. We demonstrate that spindle shape scaling is independent of the nature of the molecules that regulate dynamic microtubule properties, but is dependent on the steady-state metaphase spindle length. The shape of the spindle scales anisotropically with increasing length. Our results suggest that intrinsic mechanisms control the shape of the spindle to ensure the efficient capture and alignment of chromosomes independently of spindle length.
K-fiber; Clasp; Microtubules; Mitosis; Spindle
Protein–protein interactions are crucial for cellular homeostasis and play important roles in the dynamic execution of biological processes. While antibodies represent a well-established tool to study protein interactions of extracellular domains and secreted proteins, as well as in fixed and permeabilized cells, they usually cannot be functionally expressed in the cytoplasm of living cells. Non-immunoglobulin protein-binding scaffolds have been identified that also function intracellularly and are now being engineered for synthetic biology applications. Here we used the Designed Ankyrin Repeat Protein (DARPin) scaffold to generate binders to fluorescent proteins and used them to modify biological systems directly at the protein level. DARPins binding to GFP or mCherry were selected by ribosome display. For GFP, binders with KD as low as 160 pM were obtained, while for mCherry the best affinity was 6 nM. We then verified in cell culture their specific binding in a complex cellular environment and found an affinity cut-off in the mid-nanomolar region, above which binding is no longer detectable in the cell. Next, their binding properties were employed to change the localization of the respective fluorescent proteins within cells. Finally, we performed experiments in Drosophila melanogaster and Danio rerio and utilized these DARPins to either degrade or delocalize fluorescently tagged fusion proteins in developing organisms, and to phenocopy loss-of-function mutations. Specific protein binders can thus be selected in vitro and used to reprogram developmental systems in vivo directly at the protein level, thereby bypassing some limitations of approaches that function at the DNA or the RNA level.
DARPin; GFP; mCherry; Protein interference
iRhoms are closely related to rhomboid intramembrane proteases but lack catalytic activity. In mammals iRhoms are known to regulate the trafficking of TACE, the protease that cleaves the membrane bound inflammatory cytokine TNF. We have mapped a spontaneously occurring mouse mutation with a loss of hair phenotype, curly bare (cub), to the Rhbdf2 locus, which encodes the iRhom2 protein. The cub deletion removes the first 268 amino acids of the iRhom2 protein but is not a loss of function. We have also identified a previously reported suppressor of cub, called Mcub (modifier of curly bare), and find it to be a loss of function allele of the amphiregulin gene (Areg). Amphiregulin is an activating ligand of the epidermal growth factor receptor (EGFR) that, like TNF, is released by TACE. Our results therefore imply a regulatory link between iRhoms and EGFR signalling in mammals. We have tested the model that the cub mutation leads to iRhom2 hyperactivity and consequently excess TACE processing of amphiregulin and elevated EGFR signalling. Our results do not support this hypothesis: we find that, compared to wild-type cells, cub mutant embryonic fibroblasts release less amphiregulin, and that the cub mutant form of iRhom2 is less able than wild type to bind to TACE and promote its maturation.
Rhomboid; iRhom; Rhbdf2; Amphiregulin; Mouse; TACE; ADAM17
Mutations in the alpha-thalassemia mental retardation X-linked (ATRX) gene cause a spectrum of abnormalities including intellectual disability, developmental delay, seizures, and microcephaly. The ATRX protein is highly enriched at heterochromatic repetitive sequences adjacent to the centromere, and ATRX depletion results in chromosome congression, segregation, and cohesion defects. Here, we show that Cre-mediated inactivation of Atrx in the embryonic mouse (Mus musculus) brain results in expansion of cerebral cortical layer VI, and a concurrent thinning of layers II–IV. We observed increased cell cycle exit during early-mid neurogenesis, and a depletion of apical progenitors by late neurogenesis in the Atrx-null neocortex, explaining the disproportionate layering. Premature differentiation was associated with an increased generation of outer radial glia (oRG) and TBR2-expressing basal progenitors, as well as increased generation of early-born post-mitotic projection neurons. Atrx deletion also reduced the fidelity of mitotic spindle orientation in apical progenitors, where mutant cells were often oriented at non-parallel angles of division relative to the ventricular surface. We conclude that ATRX is required for correct lamination of the mouse neocortex by regulating the timing of neuroprogenitor cell differentiation.
ATRX; Cell division; Chromatin; Cortical lamination; Neurodevelopment
Anandamide (N-arachidonyl ethanolamide, AEA) is an endogenous cannabinoid that is involved in various pathological conditions, including cardiovascular diseases and tumor-angiogenesis. Herein, we tested the involvement of classical cannabinoid receptors (CBRs) and the Ca2+-channel transient receptor potential vanilloid 1 (TRPV1) on cellular AEA uptake and its effect on endothelial cell proliferation and network-formation. Uptake of the fluorescence-labeled anandamide (SKM4-45-1) was monitored in human endothelial colony-forming cells (ECFCs) and a human endothelial-vein cell line (EA.hy926). Involvement of the receptors during AEA translocation was determined by selective pharmacological inhibition (AM251, SR144528, CID16020046, SB366791) and molecular interference by TRPV1-selective siRNA-mediated knock-down and TRPV1 overexpression. We show that exclusively TRPV1 contributes essentially to AEA transport into endothelial cells in a Ca2+-independent manner. This TRPV1 function is a prerequisite for AEA-induced endothelial cell proliferation and network-formation. Our findings point to a so far unknown moonlighting function of TRPV1 as Ca2+-independent contributor/regulator of AEA uptake. We propose TRPV1 as representing a promising target for development of pharmacological therapies against AEA-triggered endothelial cell functions, including their stimulatory effect on tumor-angiogenesis.
transient receptor potential vanilloid 1, TRPV1; anandamide, AEA; endothelial colony-forming cells, ECFC; anandamide transport; proliferation; network-formation; angiogenesis
Vertebrate adherens junctions mediate cell–cell adhesion via a “classical” cadherin–catenin “core” complex, which is associated with and regulated by a functional network of proteins, collectively named the cadherin adhesome (“cadhesome”). The most basal metazoans have been shown to conserve the cadherin–catenin “core”, but little is known about the evolution of the cadhesome. Using a bioinformatics approach based on both sequence and structural analysis, we have traced the evolution of this larger network in 26 organisms, from the uni-cellular ancestors of metazoans, through basal metazoans, to vertebrates. Surprisingly, we show that approximately 70% of the cadhesome, including proteins with similarity to the catenins, predate metazoans. We found that the transition to multicellularity was accompanied by the appearance of a small number of adaptor proteins, and we show how these proteins may have helped to integrate pre-metazoan sub-networks via PDZ domain–peptide interactions. Finally, we found the increase in network complexity in higher metazoans to have been driven primarily by expansion of paralogs. In summary, our analysis helps to explain how the complex protein network associated with cadherin at adherens junctions first came together in the first metazoan and how it evolved into the even more complex mammalian cadhesome.
cadherin; adherens junction; multicellularity; evolution; protein interaction network
The multi-domain CX3CL1 transmembrane chemokine triggers leukocyte adherence without rolling and migration by presenting its chemokine domain (CD) to its receptor CX3CR1. Through the combination of functional adhesion assays with structural analysis using FRAP, we investigated the functional role of the other domains of CX3CL1, i.e., its mucin stalk, transmembrane domain, and cytosolic domain. Our results indicate that the CX3CL1 molecular structure is finely adapted to capture CX3CR1 in circulating cells and that each domain has a specific purpose: the mucin stalk is stiffened by its high glycosylation to present the CD away from the membrane, the transmembrane domain generates the permanent aggregation of an adequate amount of monomers to guarantee adhesion and prevent rolling, and the cytosolic domain ensures adhesive robustness by interacting with the cytoskeleton. We propose a model in which quasi-immobile CX3CL1 bundles are organized to quickly generate adhesive patches with sufficiently high strength to capture CX3CR1+ leukocytes but with sufficiently low strength to allow their patrolling behavior.
Chemokine; Adhesion; FRAP; Glycosylation; GPCR
A general view is that Schizosaccharomyces pombe undergoes symmetric cell division with two daughter cells inheriting equal shares of the content from the mother cell. Here we show that CTP synthase, a metabolic enzyme responsible for the de novo synthesis of the nucleotide CTP, can form filamentous cytoophidia in the cytoplasm and nucleus of S. pombe cells. Surprisingly, we observe that both cytoplasmic and nuclear cytoophidia are asymmetrically inherited during cell division. Our time-lapse studies suggest that cytoophidia are dynamic. Once the mother cell divides, the cytoplasmic and nuclear cytoophidia independently partition into one of the two daughter cells. Although the two daughter cells differ from one another morphologically, they possess similar chances of inheriting the cytoplasmic cytoophidium from the mother cell, suggesting that the partition of cytoophidium is a stochastic process. Our findings on asymmetric inheritance of cytoophidia in S. pombe offer an exciting opportunity to study the inheritance of metabolic enzymes in a well-studied model system.
Asymmetric inheritance; CTP synthase; Schizosaccharomyces pombe; Cytoophidium; Intracellular compartmentation
In order to gain insight into the impact of yolk increase on endoderm development, we have analyzed the mechanisms of endoderm formation in the catshark S. canicula, a species exhibiting telolecithal eggs and a distinct yolk sac. We show that in this species, endoderm markers are expressed in two distinct tissues, the deep mesenchyme, a mesenchymal population of deep blastomeres lying beneath the epithelial-like superficial layer, already specified at early blastula stages, and the involuting mesendoderm layer, which appears at the blastoderm posterior margin at the onset of gastrulation. Formation of the deep mesenchyme involves cell internalizations from the superficial layer prior to gastrulation, by a movement suggestive of ingressions. These cell movements were observed not only at the posterior margin, where massive internalizations take place prior to the start of involution, but also in the center of the blastoderm, where internalizations of single cells prevail. Like the adjacent involuting mesendoderm, the posterior deep mesenchyme expresses anterior mesendoderm markers under the control of Nodal/activin signaling. Comparisons across vertebrates support the conclusion that endoderm is specified in two distinct temporal phases in the catshark as in all major osteichthyan lineages, in line with an ancient origin of a biphasic mode of endoderm specification in gnathostomes. They also highlight unexpected similarities with amniotes, such as the occurrence of cell ingressions from the superficial layer prior to gastrulation. These similarities may correspond to homoplastic traits fixed separately in amniotes and chondrichthyans and related to the increase in egg yolk mass.
endoderm; telolecithal egg; chondrichthyan; Nodal signalling
Russian wheat aphid (Diuraphis noxia, Kurdjumov) feeding on susceptible Triticum aestivum L. leads to leaf rolling, chlorosis and plant death – symptoms not present in resistant lines. Although the effects of several D. noxia (Dn) resistance genes are known, none have been isolated or characterized. Wheat varieties expressing different Dn genes exhibit distinct modes of D. noxia resistance, such as antibiosis (Dn1), tolerance (Dn2), and antixenosis (Dn5). However, the mechanism whereby feeding aphids are perceived, and how subsequent transcriptional responses are partitioned into resistance categories, remains unclear. Here we report on downstream events in near-isogenic wheat lines containing different Dn genes after D. noxia biotype SA1 feeding. Transcripts involved in stress, signal transduction, photosynthesis, metabolism and gene regulation were differentially regulated during D. noxia feeding. Expression analyses using RT-qPCR and RNA hybridization, as well as enzyme activity profiling, provide evidence that the timing and intensity of pathways induced are critical in the development of particular modes of resistance. Pathways involved include the generation of kinase signalling cascades that lead to a sustained oxidative burst, and a hypersensitive response that is active during antibiosis. Tolerance is a passive resistance mechanism that acts through repair or de novo synthesis of photosystem proteins. Results further suggest that ethylene-mediated pathways are possibly involved in generating volatile compounds and cell wall fortification during the antixenosic response.
Aphid feeding; cDNA-AFLP; Expression profiling; Peroxidase; Glutathione-S-transferase; Lipoxygenase; β-1,3-glucanase; Affymetrix
Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by selective death of motor neurons. In 5–10% of the familial cases, the disease is inherited because of mutations. One such mutation, P56S, was identified in human VAPB that behaves in a dominant negative manner, sequestering wild type protein into cytoplasmic inclusions.
We have conducted a reverse genetic screen to identify interactors of Drosophila VAPB. We screened 2635 genes and identified 103 interactors, of which 45 were enhancers and 58 were suppressors of VAPB function. Interestingly, the screen identified known ALS loci – TBPH, alsin2 and SOD1. Also identified were genes involved in cellular energetics and homeostasis which were used to build a gene regulatory network of VAPB modifiers. One key modifier identified was Tor, whose knockdown reversed the large bouton phenotype associated with VAP(P58S) expression in neurons. A similar reversal was seen by over-expressing Tuberous Sclerosis Complex (Tsc1,2) that negatively regulates TOR signaling as also by reduction of S6K activity. In comparison, the small bouton phenotype associated with VAP(wt) expression was reversed with Tsc1 knock down as well as S6K-CA expression. Tor therefore interacts with both VAP(wt) and VAP(P58S), but in a contrasting manner. Reversal of VAP(P58S) bouton phenotypes in larvae fed with the TOR inhibitor Rapamycin suggests upregulation of TOR signaling in response to VAP(P58S) expression.
The VAPB network and further mechanistic understanding of interactions with key pathways, such as the TOR cassette, will pave the way for a better understanding of the mechanisms of onset and progression of motor neuron disease.
VAP; Neurodegeneration; TOR; ALS; Drosophila RNAi screen
In molting animals, a cuticular extracellular matrix forms the first barrier to infection and other environmental insults. In the nematode Caenorhabditis elegans there are two types of cuticle: a well-studied collagenous cuticle lines the body, and a poorly-understood chitinous cuticle lines the pharynx. In the posterior end of the pharynx is the grinder, a tooth-like cuticular specialization that crushes food prior to transport to the intestine for digestion. We here show that the grinder increases in size only during the molt. To gain molecular insight into the structure of the grinder and pharyngeal cuticle, we performed a microarray analysis to identify mRNAs increased during the molt. We found strong transcriptional induction during the molt of 12 of 15 previously identified abu genes encoding Prion-like (P) glutamine (Q) and asparagine (N) rich PQN proteins, as well as 15 additional genes encoding closely related PQN proteins. abu/pqn genes, which we name the abu/pqn paralog group (APPG) genes, were expressed in pharyngeal cells and the proteins encoded by two APPG genes we tested localized to the pharyngeal cuticle. Deleting the APPG gene abu-14 caused abnormal pharyngeal cuticular structures and knocking down other APPG genes resulted in abnormal cuticular function. We propose that APPG proteins promote the assembly and function of a unique cuticular structure. The strong developmental regulation of the APPG genes raises the possibility that such genes would be identified in transcriptional profiling experiments in which the animals' developmental stage is not precisely staged.
C. elegans; molting; larvae; amyloid; cuticle; ABU/PQN; innate immunity; unfolded protein response
Rab GTPases are master regulators of eukaryotic endomembrane systems, particularly functioning in membrane tethering to confer the directionality of intracellular membrane trafficking. However, how exactly Rab GTPases themselves act upon membrane tethering processes has remained enigmatic. Here, we thoroughly tested seven purified Rab GTPases in human, which localize at the various representative organelles, for their capacity to support membrane tethering in vitro. Strikingly, we found that three specific human Rabs (endoplasmic reticulum/Golgi Rab2a, early endosomal Rab5a, and late endosomal/lysosomal Rab7a) strongly accelerated membrane aggregation of synthetic liposomes even in the absence of any additional components, such as classical tethers, tethering factors, and Rab effectors. This Rab-induced membrane aggregation was a reversible membrane tethering reaction that can be strictly controlled by the membrane recruitment of Rab proteins on both apposing membranes. Thus, our current reconstitution studies establish that membrane-anchored human Rab GTPases are an essential tethering factor to directly mediate membrane tethering events.
Rab GTPase; Liposome; Membrane tethering; Membrane traffic; Reconstitution
The essential metabolic enzyme CTP synthase (CTPsyn) can be compartmentalised to form an evolutionarily-conserved intracellular structure termed the cytoophidium. Recently, it has been demonstrated that the enzymatic activity of CTPsyn is attenuated by incorporation into cytoophidia in bacteria and yeast cells. Here we demonstrate that CTPsyn is regulated in a similar manner in Drosophila tissues in vivo. We show that cytoophidium formation occurs during nutrient deprivation in cultured cells, as well as in quiescent and starved neuroblasts of the Drosophila larval central nervous system. We also show that cytoophidia formation is reversible during neurogenesis, indicating that filament formation regulates pyrimidine synthesis in a normal developmental context. Furthermore, our global metabolic profiling demonstrates that CTPsyn overexpression does not significantly alter CTPsyn-related enzymatic activity, suggesting that cytoophidium formation facilitates metabolic stabilisation. In addition, we show that overexpression of CTPsyn only results in moderate increase of CTP pool in human stable cell lines. Together, our study provides experimental evidence, and a mathematical model, for the hypothesis that inactive CTPsyn is incorporated into cytoophidia.
CTP synthase; cytoophidium; intracellular compartmentation; CTP; Drosophila; neurogenesis
Intracellular trafficking of viruses and proteins commonly occurs via the early endosome in a process involving Rab5. The RNA Import Complex (RIC)-RNA complex is taken up by mammalian cells and targeted to mitochondria. Through RNA interference, it was shown that mito-targeting of the ribonucleoprotein (RNP) was dependent on caveolin 1 (Cav1), dynamin 2, Filamin A and NSF. Although a minor fraction of the RNP was transported to endosomes in a Rab5-dependent manner, mito-targeting was independent of Rab5 or other endosomal proteins, suggesting that endosomal uptake and mito-targeting occur independently. Sequential immunoprecipitation of the cytosolic vesicles showed the sorting of the RNP away from Cav1 in a process that was independent of the endosomal effector EEA1 but sensitive to nocodazole. However, the RNP was in two types of vesicle with or without Cav1, with membrane-bound, asymmetrically orientated RIC and entrapped RNA, but no endosomal components, suggesting vesicular sorting rather than escape of free RNP from endosomes. In vitro, RNP was directly transferred from the Type 2 vesicles to mitochondria. Live-cell imaging captured spherical Cav1− RNP vesicles emerging from the fission of large Cav+ particles. Thus, RNP appears to traffic by a different route than the classical Rab5-dependent pathway of viral transport.
RNA protein complex; Endosome; Sorting; Caveolin 1; Mitochondria
Skeletal muscle fibre type, cross-sectional area (CSA), maximum enzyme capacities and fibre oxidative capacities were investigated in three southern African antelope species. Muscle samples from blesbok (Damaliscus pygargus phillipsi), mountain reedbuck (Redunca fulvorufula) and greater kudu (Tragelaphus strepsiceros) were collected post mortem from the Vastus lateralis and analysed for myosin heavy chain (MHC) isoform content, citrate synthase (CS), 3-hydroxyacyl Co A dehydrogenase (3-HAD), phosphofructokinase (PFK), lactate dehydrogenase (LDH) and creatine kinase (CK) activities. Histochemistry and immunohistochemistry were performed to determine relative fibre oxidative capacity, fibre type and cross-sectional area (CSA). Type IIX fibres were the most abundant fibre type in all three species, ranging from 43 to 57%. Kudu had less type IIX fibres than mountain reedbuck and blesbok (P<0.05), values confirmed by their respective MHC isoform content. Blesbok had the smallest fibres, followed by mountain reedbuck and finally kudu (P<0.001). Overall, all three species had high oxidative and glycolytic capacities, but species differences were found. Kudu had the lowest CS activity, followed by blesbok and mountain reedbuck, but the highest PFK, LDH and CK activities. This study confirmed large variation in oxidative capacities within a single fibre type, as well as overlap between the fibre types with no distinct differences between the three species. The fibre type profile of each species is discussed and confirms some of their physical attributes and capabilities.
Wild animals; Enzymes; Cross-sectional area; Fibre type; Metabolism
Take-off in bats is separated into two distinct phases: an initial jump and a subsequent wing powered acceleration. Here, using footage from a high-speed camera, the first comparative study of the performance during the wing induced phase of take-off in six insectivorous bat species is described. Despite distinct differences in foraging strategy, the mass specific power generated by the bats during wing induced take-off did not differ between species, with the exception of Myotis capaccinii. This suggests that differences in take-off performance may only be evident in bats that exhibit particularly unusual foraging strategies, such as the trawling behaviour of M. capaccinii – with differences in the remaining species only manifesting in subtler aspects of flight performance such as agility or manoeuvrability. The poorer take-off performance of M. capaccinii could be related to either a reduction in wing-stroke amplitude to stop the wings hitting the water's surface during foraging or perhaps an effect of having very large feet. No scaling relationship between body mass and mass-specific take-off power was found, which supports earlier research on birds and insects, suggesting that the mass-specific muscle power available for flight is broadly similar across a large range of body sizes and species.
biomechanics; jumping; muscle; scaling; take-off; bat
Carbon dioxide (CO2) levels projected to occur in the oceans by the end of this century cause a range of behavioural effects in fish, but whether other highly active marine organisms, such as cephalopods, are similarly affected is unknown. We tested the effects of projected future CO2 levels (626 and 956 µatm) on the behaviour of male two-toned pygmy squid, Idiosepius pygmaeus. Exposure to elevated CO2 increased the number of active individuals by 19–25% and increased movement (number of line-crosses) by nearly 3 times compared to squid at present-day CO2. Squid vigilance and defensive behaviours were also altered by elevated CO2 with >80% of individuals choosing jet escape responses over defensive arm postures in response to a visual startle stimulus, compared with 50% choosing jet escape responses at control CO2. In addition, more escape responses were chosen over threat behaviours in body pattern displays at elevated CO2 and individuals were more than twice as likely to use ink as a defence strategy at 956 µatm CO2, compared with controls. Increased activity could lead to adverse effects on energy budgets as well as increasing visibility to predators. A tendency to respond to a stimulus with escape behaviours could increase survival, but may also be energetically costly and could potentially lead to more chases by predators compared with individuals that use defensive postures. These results demonstrate that projected future ocean acidification affects the behaviours of a tropical squid species.
Ocean acidification; Cephalopod; Anti-predator behaviour; Escape; Avoidance; Startle response