Biology subjects

Brownlee, C.

Publications and source records attributed to Brownlee, C..

14 recordsLinked to original sources

bioRxiv2024

Identification of a motif in TPX2 that regulates spindle architecture in Xenopus egg extracts

A bipolar spindle composed of microtubules and many associated proteins functions to segregate chromosomes during cell division in all eukaryotes, yet spindle size and architecture varies dramatically across different species and cell types. Targeting protein for Xklp2 (TPX2) is one candidate factor for modulating spindle microtubule organization through its roles in branching microtubule nucleation, activation of the mitotic kinase Aurora A, and association with the kinesin-5 (Eg5) motor. Here we identify a conserved nuclear localization sequence (NLS) motif, 123KKLK126 in X. laevis TPX2, which regulates astral microtubule formation and spindle pole morphology in Xenopus egg extracts. Addition of recombinant TPX2 with this sequence mutated to AALA dramatically increased spontaneous formation of microtubule asters and recruitment of phosphorylated Aurora A, pericentrin, and Eg5 to meiotic spindle poles. We propose that TPX2 is a linchpin spindle assembly factor whose regulation contributes to the recruitment and activation of multiple microtubule polymerizing and organizing proteins, generating distinct spindle architectures.

cell biology↗

bioRxiv2023

Plant-like heliotropism in a photosymbiotic animal

Being photosynthetic sessile organisms, plants established heliotropism to track the suns position across the sky and allow their vegetative parts to orient accordingly. Here, we report plant-like heliotropic movement in a photosymbiotic sea anemone Anemonia viridis. Like plants, photosynthesis represents a key energy source in endosymbiotic cnidarians bearing microalgae. We observed that A. viridis in their natural habitats under sunlight displayed heliotropism or solar tracking by pointing their tentacles towards the sun while remaining sessile, facing east at dawn and west at dusk as they track the suns relative position through the day, a phenomenon previously only observed in plants. Solar tracking movements in A. viridis are driven by a light wavelength that prompts photosynthesis in their endosymbionts. The heliotropic response was absent in both bleached (aposymbiotic) A. viridis and in symbiotic A. viridis with chemically inhibited photosynthesis. We revealed a direct correlation between heliotropism and endosymbiont oxygen production in A. viridis. Our findings suggest that photosymbiotic A. viridis has likely evolved plant-like heliotropism as an effective way to modulate exposure to solar irradiation for photosynthesis. The study exemplifies how photosynthetic organisms such as plants and photosymbiotic sea anemones, display similar behaviour in response to similar environmental pressures.

animal behavior and cognition↗

bioRxiv2023

Diatoms exhibit dynamic chloroplast calcium signals in response to high light and oxidative stress

Diatoms are a group of silicified algae that play a major role in marine and freshwater ecosystems. Diatom chloroplasts were acquired by secondary endosymbiosis and exhibit important structural and functional differences from the primary plastids of land plants and green algae. Many functions of primary plastids, including photoacclimation and inorganic carbon acquisition, are regulated by calcium-dependent signalling processes. Calcium signalling has also been implicated in the photoprotective responses of diatoms, although the nature of calcium elevations in diatom chloroplasts and their wider role in cell signalling remains unknown. Using genetically encoded calcium indicators, we find that the diatom Phaeodactylum tricornutum exhibits dynamic chloroplast calcium elevations that are distinct from those found in land plants. Chloroplast calcium ([Ca2+]chl) acts independently from the cytosol and is not elevated by stimuli that induce large cytosolic calcium ([Ca2+]cyt) elevations. In contrast, high light and exogenous hydrogen peroxide (H2O2) induce large, sustained calcium elevations in the chloroplast stroma that are not replicated in the cytosol. Measurements using the fluorescent H2O2 sensor roGFP2-Orp1 indicate that [Ca2+]chl elevations induced by these stimuli correspond to the accumulation of H2O2 in the chloroplast. [Ca2+]chl elevations were also induced by the addition of methyl viologen, which acts to generate superoxide within the chloroplast, and by treatments that disrupt non-photochemical quenching (NPQ). The findings indicate that diatoms generate specific calcium elevations in the chloroplast in response to high light and oxidative stress that likely modulate the activity of calcium-sensitive components in photoprotection and other regulatory pathways.

plant biology↗

bioRxiv2023

Cell Type-Specific Regulation by a Heptad of Transcription Factors in Human Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay of transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs - FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2 - bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remained unknown. We mapped genome-wide chromatin contacts and TF binding profiles in HSPC subsets (HSC, CMP, GMP, MEP) and found that heptad occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. These findings suggest that specific heptad-TF combinations play critical roles in regulating hematopoietic differentiation and provide a valuable resource for development of targeted therapies to manipulate specific HSPC subsets.

developmental biology↗

bioRxiv2023

The intracellular lifestyle of an archaeal symbiont

DPANN Archaea are a diverse group of organisms typically characterised by small cells and reduced genomes. To date, all cultivated DPANN Archaea are ectosymbionts that require direct cell contact with an archaeal host species for proliferation. However, the dynamics of DPANN - host interactions and the impacts of these interactions on the host species are poorly understood. Here, we show that one DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. Our data also suggest that these interactions involve invasion of the host cell by the nanohaloarchaeon. This is the first reported instance of such a predatory-like lifestyle amongst Archaea and indicates that some DPANN Archaea may interact with host populations in a manner similar to viruses.

microbiology↗

bioRxiv2022

Cold-induced cyt elevations function to support osmoregulation in marine diatoms

Diatoms are a group of microalgae that are important primary producers in a range of open ocean, freshwater and intertidal environments. The latter can experience significant long- and short-term variability in temperature, from seasonal variations to rapid temperature shifts caused by tidal immersion and emersion. As temperature is a major determinant in the distribution of diatom species, their temperature sensory and response mechanisms likely have important roles in their ecological success. We have examined the mechanisms diatoms use to sense rapid changes in temperature, such as those experienced in the intertidal zone. We find that the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana exhibit a transient cytosolic Ca2+ ([Ca2+]cyt) elevation in response to rapid cooling, similar to those observed in plant and animal cells. However, [Ca2+]cyt elevations were not observed in response to rapid warming. The kinetics and magnitude of cold-induced [Ca2+]cyt elevations correlate with the rate of temperature decrease. We do not find a role for the [Ca2+]cyt elevations in enhancing cold tolerance, but show that cold shock induces a Ca2+-dependent K+ efflux and reduces mortality of P. tricornutum during a simultaneous hypo-osmotic shock. As inter-tidal diatom species may routinely encounter simultaneous cold and hypo-osmotic shocks during tidal cycles, we propose that cold-induced Ca2+ signalling interacts with osmotic signalling pathways to aid in the regulation of cell volume. Our findings provide insight into the nature of temperature perception in diatoms and highlight that cross-talk between signalling pathways may play an important role in their cellular responses to multiple simultaneous stressors.

plant biology↗

bioRxiv2022

The effect of cytoskeleton inhibitors on coccolith morphology in Coccolithus braarudii and Scyphosphaera apsteinii

The calcite platelets of coccolithophores (Haptophyta), the coccoliths, are among the most elaborate biomineral structures. How these unicellular algae accomplish the complex morphogenesis of coccoliths is still largely unknown. It has long been proposed that the cytoskeleton plays a central role in shaping the growing coccoliths. Previous studies have indicated that disruption of the microtubule network led to defects in coccolith morphogenesis in Emiliania huxleyi and Coccolithus braarudii. Disruption of the actin network also led to defects in coccolith morphology in E. huxleyi, but its impact on coccolith morphology in C. braarudii was less clear, as coccolith secretion was largely inhibited under the conditions used. A more detailed examination of the role of actin and microtubule networks is therefore required to address the wider role of the cytoskeleton in coccolith morphogenesis. In this study, we have examined coccolith morphology in C. braarudii and Scyphosphaera apsteinii following treatment with the microtubule inhibitors vinblastine and colchicine (S. apsteinii only) and the actin inhibitor cytochalasin B. We found that all cytoskeleton inhibitors induced coccolith malformations, strongly suggesting that both microtubules and actin filaments are instrumental in morphogenesis. By demonstrating the requirement for the microtubule and actin networks in coccolith morphogenesis in diverse species, our results suggest that both of these cytoskeletal elements are likely to play conserved roles in defining coccolith morphology.

microbiology↗

bioRxiv2022

Distinct physiological responses of Coccolithus braarudii life cycle phases to light intensity and nutrient availability

Coccolithophores feature a haplo-diplontic life cycle comprised of diploid cells producing heterococcoliths and haploid cells producing morphologically different holococcoliths. These life cycle phases of each species appear to have distinct spatial and temporal distributions in the oceans, with the heavily-calcified heterococcolithophores (HET) often more prevalent in winter and at greater depths, whilst the lightly-calcified holococcolithophores (HOL) are more abundant in summer and in shallower waters. The haplo-diplontic life cycle may therefore allow coccolithophores to expand their ecological niche, switching between life cycle phases to exploit conditions that are more favourable. However, coccolithophore life cycles remain poorly understood and fundamental information on the physiological differences between life cycle phases is required if we are to better understand the ecophysiology of coccolithophores. In this study, we have examined the physiology of HET and HOL phases of the coccolithophore Coccolithus braarudii in response to changes in light and nutrient availability. We found that the HOL phase was more tolerant to high light than the HET phase, which exhibited defects in calcification at high irradiances. The HET phase exhibited defects in coccolith formation under both nitrate (N) and phosphate (P) limitation, whilst no defects in calcification were detected in the HOL phase. The HOL phase grew to a higher cell density under P-limitation than N-limitation, whereas no difference was observed in the maximum cell density reached by the HET phase at these nutrient concentrations. HET cells grown under a light:dark cycle divided primarily in the dark and early part of the light phase, whereas HOL cells continued to divide throughout the 24 h period. The physiological differences may contribute to the distinct biogeographical distributions observed between life cycle phases, with the HOL phase potentially better adapted to high light, low nutrient regimes, such as those found in seasonally stratified surface waters. HighlightsO_LICoccolithus braarudii life cycle phases exhibit different physiological responses. C_LIO_LIThe heavily-calcified heterococcolithophores (HET) life cycle phase is more sensitive to high light. C_LIO_LIThe lightly-calcified holococcolithophores (HOL) life cycle phase may be better suited to growth under low phosphate availability. C_LI

microbiology↗

bioRxiv2021

Reduced H+ channel activity disrupts pH homeostasis and calcification in coccolithophores at low ocean pH

Coccolithophores produce the bulk of ocean biogenic calcium carbonate but this process is predicted to be negatively affected by future ocean acidification scenarios. Since coccolithophores calcify intracellularly, the mechanisms through which changes in seawater carbonate chemistry affect calcification remain unclear. Here we show that voltage-gated H+ channels in the plasma membrane of Coccolithus braarudii serve to regulate pH and maintain calcification under normal conditions, but have greatly reduced activity in cells acclimated to low pH. This disrupts intracellular pH homeostasis and impairs the ability of C. braarudii to remove H+ generated by the calcification process, leading to specific coccolith malformations. These coccolith malformations can be reproduced by pharmacological inhibition of H+ channels. Heavily-calcified coccolithophore species such as C. braarudii, which make the major contribution to carbonate export to the deep ocean, have a large intracellular H+ load and are likely to be most vulnerable to future decreases in ocean pH.

microbiology↗

bioRxiv2021

Mesoderm-Derived PDGFRA+ Cells Regulate the Emergence of Hematopoietic Stem Cells in the Dorsal Aorta

Mouse hematopoietic stem cells (HSCs) first emerge at embryonic day 10.5 (E10.5) on the ventral surface of the dorsal aorta, by endothelial-to-hematopoietic transition (EHT). We investigated whether cells with mesenchymal stem cell-like activity, which provide an essential niche for long-term HSCs (LT-HSCs) in the bone marrow, reside in the aorta- gonad-mesonephros (AGM) and contribute to the structural development of the dorsal aorta and EHT. Using transgenic mice, we demonstrate a lineage hierarchy for AGM stromal cells and traced the E10.5/E11.5 aortic endothelium and HSCs to mesoderm derived (Mesp1) PDGFRA+ stromal cells (Mesp1der PSCs). Mesp1der PSCs dominate the sub-endothelial and ventral stroma in the E10.5-E11.5 AGM but by E13.5 were replaced by neural crest (Wnt1) derived PDGFRA+ stromal cells (Wnt1der PSCs). Co-aggregating non-hemogenic embryonic and adult endothelial cells with Mesp1der PSCs but not with Wnt1der PSCs resulted in activation of a hematopoietic transcriptional program in endothelial cells accompanied by EHT and generation of LT-HSCs. Dose-dependent inhibition of PDGFRA signalling or BMP, WNT, NOTCH signalling interrupted this reprogramming event. This partnership between endothelial cells and AGM Mesp1der PSCs could potentially be harnessed to manufacture LT-HSCs from endothelium.

developmental biology↗

bioRxiv2020

Label-free Isolation and Single Cell Biophysical Phenotyping Analysis of Primary Cardiomyocytes using Inertial Microfluidics

To advance our understanding of cardiomyocyte identity and function, we need appropriate tools to isolate pure primary cardiomyocytes. We have developed a label-free method to purify viable cardiomyocytes from mouse neonatal hearts using a simple inertial microfluidics biochip. Cardiomyocytes were sorted from neonatal hearts and isolated to >90% purity and their physico-mechanical properties were evaluated using real time deformability cytometry. Purified cardiomyocytes were viable and retained their identity and function as depicted by expression of cardiac specific markers and contractility. Furthermore, we showed that cardiomyocytes have a distinct physico-mechanical phenotype that could be used as an intrinsic biophysical marker to distinguish these cells from other cell types within the heart. Taken together, this cardiomyocyte isolation and phenotyping method could serve as a valuable tool to progress our understanding of cardiomyocyte identity and function, which will ultimately benefit many diagnostic development and cardiac treatment studies.

bioengineering↗

bioRxiv2020

Flagella Ca2+ elevations regulate pausing of retrograde intraflagellar transport trains in adherent Chlamydomonas flagella

The movement of ciliary membrane proteins is directed by transient interactions with intraflagellar transport (IFT) trains. The green alga Chlamydomonas has adapted this process for gliding motility, using IFT to move adhesive glycoproteins (FMG-1B) in the flagella membrane. Although Ca2+ signalling contributes directly to the gliding process, uncertainty remains over the mechanisms through which Ca2+ acts to influence the movement of IFT trains. Here we show that flagella Ca2+ elevations regulate IFT primarily by initiating the movement of paused retrograde IFT trains. Flagella Ca2+ elevations exhibit complex spatial and temporal properties, including high frequency repetitive Ca2+ elevations that prevent the accumulation of paused retrograde IFT trains. We show that flagella Ca2+ elevations disrupt the IFT-dependent movement of microspheres along the flagella membrane. The results suggest that flagella Ca2+ elevations directly disrupt the interaction between retrograde IFT particles and flagella membrane glycoproteins to modulate gliding motility and the adhesion of the flagellum to a surface.

cell biology↗

bioRxiv2020

A novel Ca2+ signalling pathway co-ordinates environmental phosphorus sensing and nitrogen metabolism in marine diatoms

Diatoms are a diverse and globally important phytoplankton group, responsible for an estimated 20% of carbon fixation on Earth. They frequently form spatially extensive phytoplankton blooms, responding rapidly to increased availability of nutrients including phosphorus and nitrogen. Although it is well established that diatoms are common first-responders to nutrient influxes in aquatic ecosystems, little is known of the sensory mechanisms that they employ for nutrient perception. Here we show that diatoms use a novel and highly-sensitive Ca2+-dependent signalling pathway, not previously described in eukaryotes, to sense and respond to the critical macronutrient phosphorus. We demonstrate that phosphorus-Ca2+ signalling is essential for regulating diatom recovery from phosphorus limitation, by controlling rapid and substantial increases in nitrogen assimilation. Phosphorus-Ca2+ signalling thus mediates fundamental cross-talk between the vital nutrients P and N to maximise resource competition, and likely governs the success of diatoms as major bloom formers in regions of pulsed nutrient supply. Importantly, our study demonstrates that distinct mechanisms for nutrient sensing have evolved in photosynthetic eukaryotes.

microbiology↗

bioRxiv2020

A novel single-domain Na+-selective voltage-gated channel in photosynthetic eukaryotes

The evolution of Na+-selective four-domain voltage-gated channels (4D-Navs) in animals allowed rapid Na+-dependent electrical excitability, and enabled the development of sophisticated systems for rapid and long-range signalling. Whilst bacteria encode single-domain Na+-selective voltage-gated channels (BacNav), they typically exhibit much slower kinetics than 4D-Navs, and are not thought to have crossed the prokaryote-eukaryote boundary. As such, the capacity for rapid Na+-selective signalling is considered to be confined to certain animal taxa, and absent from photosynthetic eukaryotes. Certainly, in land plants, such as the Venus Flytrap where fast electrical excitability has been described, this is most likely based on fast anion channels. Here, we report a unique class of eukaryotic Na+-selective single-domain channels (EukCatBs) that are present primarily in haptophyte algae, including the ecologically important calcifying coccolithophores. The EukCatB channels exhibit very rapid voltage-dependent activation and inactivation kinetics, and sensitivity to the highly selective 4D-Nav blocker tetrodotoxin. The results demonstrate that the capacity for rapid Na+-based signalling in eukaryotes is not restricted to animals or to the presence of 4D-Navs. The EukCatB channels therefore represent an independent evolution of fast Na+-based electrical signalling in eukaryotes that likely contribute to sophisticated cellular control mechanisms operating on very short time scales in unicellular algae. One Sentence SummaryThe capacity for rapid Na+-based signalling has evolved in ecologically important coccolithophore species via a novel class of voltage-gated Na+ channels, EukCatBs.

microbiology↗