Biology subjects

Gallagher, M. T.

Publications and source records attributed to Gallagher, M. T..

5 recordsLinked to original sources

bioRxiv2025

The RNA splicing factor PRPF8 is required for left-right organiser cilia function and determination of cardiac left-right asymmetry via regulation of Arl13b splicing

Cilia function in the left-right organizer (LRO) is critical for determining internal organ asymmetry in vertebrates. To further understand the genetics of left-right asymmetry, we isolated a mouse mutant with laterality defects, l11Jus27, from a random mutagenesis screen. l11Jus27 mutants carry a missense mutation in the pre-mRNA processing factor, Prpf8. cephaloph[o]nus (cph) mutant zebrafish, carrying a protein truncating mutation in prpf8, phenocopy the laterality defects of l11Jus27 mutants. Prpf8 mutant mouse and fish embryos have increased expression of an alternative transcript encoding the cilium-associated protein, ARL13B, that lacks exon 9. In zebrafish, over-expression of the arl13b transcript lacking exon 9 perturbed cilium formation and caused laterality defects. The shorter ARL13B protein isoform lacked interactions with intraflagellar transport proteins. Our data suggest that PRPF8 plays a prominent role in LRO cilia by through the regulation of alternative splicing of ARL13B, thus uncovering a new mechanism for cilia-linked developmental defects.

developmental biology↗

bioRxiv2022

Axonemal regulation by curvature explains sperm flagellar waveform modulation

Flagellar motility is critical to natural and many forms of assisted reproduction. The rhythmic beating and wave propagation by the flagellum propels sperm through fluid and enables modulation between penetrative progressive motion, activated side-to-side yaw and hyperactivated movement associated with detachment from epithelial binding. These changes occur in response to the properties of the surrounding fluid environment, biochemical activation state, and physiological ligands, however a parsimonious mechanistic explanation of flagellar beat generation that can explain motility modulation is lacking. In this paper we present the Axonemal Regulation of Curvature, Hysteretic model (ARCH), a curvature control-type model based on switching of active moment by local curvature, embedded within a geometrically nonlinear elastic model of the flagellum exhibiting planar flagellar beats, together with nonlocal viscous fluid dynamics. The biophysical system is parameterised completely by four dimensionless parameter groupings. The effect of parameter variation is explored through computational simulation, revealing beat patterns that are qualitatively representative of penetrative (straight progressive), activated (highly yawing) and hyperactivated (non-progressive) modes. Analysis of the flagellar limit cycles and associated swimming velocity reveals a cusp catastrophe between progressive and non-progressive modes, and hysteresis in the response to changes in critical curvature parameter. Quantitative comparison to experimental data on human sperm exhibiting typical penetrative, activated and hyperactivated beats shows a good fit to the time-average absolute curvature profile along the flagellum, providing evidence that the model is capable of providing a framework for quantitative interpretation of imaging data.

cell biology↗

bioRxiv2022

The ribosome inhibitor chloramphenicol induces motility deficits in human spermatozoa: a proteomic approach identifies potentially involved proteins

Mature spermatozoa are almost completely devoid of cytoplasm; as such it has long been believed that they do not contain ribosomes and are therefore not capable of synthesising proteins. However, since the 1950s, various studies have shown translational activity within spermatozoa, particularly during their in vitro capacitation. Most of them demonstrated that mitochondrial (and not cytoplasmic) ribosomes would be involved in the translation of mitochondrial and nuclear-encoded cytoplasmic mRNAs. However, some evidence suggests that cytoplasmic ribosomes could also be active. Here, we investigate the presence and activity of the two types of ribosomes in mature human spermatozoa. By targeting ribosomal RNAs and proteins, we show that both types of ribosomes are localized in the midpiece as well as in the neck and the base of the head of the spermatozoa. We assessed the impact of cycloheximide (CHX) and chloramphenicol (CP), inhibitors of cytoplasmic and mitochondrial ribosomes, respectively, on different sperm parameters. Neither CHX, nor CP impacted sperm vitality, mitochondrial activity (measured through the ATP content), or capacitation (measured through the content in phosphotyrosines). However, increasing CP concentrations induced a decrease in total and progressive motilities as well as on some kinematic parameters while no effect was observed with CHX. A quantitative proteomic analysis was performed by mass spectrometry in SWATH mode to compare the proteomes of spermatozoa capacitated in the absence or presence of the two ribosome inhibitors. Among the [~] 700 proteins identified in the different tested conditions, 3, 3 and 25 proteins presented a modified abundance in the presence of 1 and 2 mg/ml of CHX, and 1 mg/ml of CP, respectively. The observed abundance variations of some CP-down regulated proteins were validated using Multiple-Reaction Monitoring (MRM). Taken together, our results show that the sperm motility deficits induced in the presence of CP could be linked to the observed decrease of the abundance of several proteins, at least FUNDC2 and QRICH2.

cell biology↗

bioRxiv2019

Simulations of particle tracking in the oligociliated mouse node and implications for left-right symmetry breaking mechanics

The concept of internal anatomical asymmetry is familiar; usually in humans the heart is on the left and the liver is on the right, however how does the developing embryo know to produce this consistent laterality? Symmetry breaking initiates with left-right asymmetric cilia-driven fluid mechanics in a small fluid-filled structure called the ventral node in mice. However the question of what converts this flow into left-right asymmetric development remains unanswered. A leading hypotheses is that flow transports morphogen containing vesicles within the node, the absorption of which results in asymmetrical gene expression. To investigate how vesicle transport might result in the situs patterns observed in wildtype and mutant experiments, we extend the open source Stokes flow package, NEAREST, to consider the hydrodynamic and Brownian motion of particles in a mouse model with flow driven by one, two, and 112 beating cilia.Three models for morphogen-containing particle released are simulated to assess their compatibility with observed results in oligociliated and wildtype mouse embryos: uniformly random release, localised cilium stress induced release, and localised release from motile cilia themselves. Only the uniformly random release model appears consistent with the data, with neither localised-release model resulting in significant transport in the oligociliated embryo.

developmental biology↗

bioRxiv2019

Rapid sperm capture: High-throughput flagellar waveform analysis

Flagella are critical across all eukaryotic life, and the human sperm flagellum is crucial to natural fertility. Existing automated sperm diagnostics (CASA) rely on tracking the sperm head and extrapolating measures. We describe fully-automated tracking and analysis of flagellar movement for large cell numbers. The analysis is demonstrated on freely-motile cells in low and high viscosity fluids, and validated on published data of tethered cells undergoing pharmacological hyperactivation. Direct analysis of the flagellar beat reveals that the CASA measure beat cross frequency, does not measure beat frequency. A new measurement, track centroid speed, is validated as an accurate differentiator of progressive motility. Coupled with fluid mechanics codes, waveform data enables extraction of experimentally intractable quantities such as energy dissipation, disturbance of the surrounding medium and viscous stresses. We provide a powerful and accessible research tool, enabling connection of the cells mechanical activity to its motility and effect on its environment.

cell biology↗