Biology subjects

Paschke, P.

Publications and source records attributed to Paschke, P..

6 recordsLinked to original sources

bioRxiv2026

A conserved mechanism for regulation of mtDNA copy number in eukaryotes

Mitochondrial mass and mitochondrial DNA (mtDNA) copy number are coupled to metabolic demand at the cellular, tissue and organismal level, however, the molecular basis for homeostatic regulation of mtDNA is not understood. Here we show that mitochondria and mtDNA copy number are regulated by compartmentalisation of iron-sulfur (Fe-S) clusters, glutathione and cysteine, a mechanism we exemplify in model systems ranging from plants to human cells. Using genome-wide CRISPR screens we discovered that the mitochondrial ABC-family transporter, ABCB7, is a negative regulator of mtDNA copy number. Partial silencing of ABCB7 in human cells increased mtDNA 2-3 fold, enhancing mitochondrial mass and function. ABCB7 silencing compelled co-incident mitochondrial accumulation and cytosolic depletion of Fe-S clusters, simultaneously engaging the cellular iron starvation response and stabilising the mitochondrial glutathione transporter, SLC25A39. Transport of glutathione from the cytosol into mitochondria was co-incident with mitochondrial cysteine accumulation and cytosolic cysteine depletion, which was necessary and sufficient to increase mtDNA copy number in an integrated stress response-dependent fashion, with induction of PGC1{beta} and ERR. Silencing or partial loss of function mutations in the ABCB7 homologs of D.melanogaster, S.cerevisiae and A. thaliana elicited similar increases of mtDNA within these organisms. These data reveal a fundamental metabolic logic coupling compartmentalisation of redox co-factors to organellar genome content; a conserved axis across eukaryotes that pre-dates several elements of the mtDNA replication machinery.

cell biology↗

bioRxiv2022

Competition between chemoattractants causes unexpected complexity and can explain negative chemotaxis

Negative chemotaxis, where eukaryotic cells migrate away from repellents, is important throughout biology, for example in nervous system patterning and resolution of inflammation. However, the mechanisms by which molecules repel migrating cells are unknown. Here, we use a combination of modelling and experiments with Dictyostelium cells to show that competition between different ligands that bind to the same receptor leads to effective chemorepulsion. 8-CPT-cAMP, widely described as a simple chemorepellent, is inactive on its own, and only repels cells if it interacts with the attractant cAMP. If cells degrade either competing ligand, the pattern of migration becomes more complex; cells may be repelled in one part of a gradient but attracted elsewhere, leading to populations moving in different directions in the same assay, or converging in an arbitrary place. More counterintuitively still, two chemicals can each attract cells on their own, but repel cells when combined together. We have thus identified a new mechanism that drives reverse chemotaxis, verified by mathematical models and experiments with real cells, and important anywhere several ligands compete for the same receptors.

cell biology↗

bioRxiv2022

The formation and closure of macropinocytic cups in a model system

Macropinocytosis is a conserved endocytic process where cells take up medium into micron-sized vesicles. In Dictyostelium, macropinocytic cups form around domains of PIP3 in the plasma membrane and extend by actin polymerization. Using lattice light-sheet microscopy, we describe how cups originate, are supported by an F-actin scaffold and shaped by a ring of actin polymerization, created around PIP3 domains. How cups close is unknown. We find two ways: lip closure, where actin polymerization at the lip is re-directed inwards; and basal closure, where it stretches the cup, eventually causing membrane delamination and vesicle sealing. Cups grow as expanding waves of actin polymerization that travel across the cell surface, capturing new membrane. We propose that cups close when these waves stall. This stalled wave hypothesis is tested through a conceptual model, where the interplay of forces from actin polymerization and membrane tension recreates many of our observations.

cell biology↗

bioRxiv2022

The Scar/WAVE complex drives normal actin protrusions without the Arp2/3 complex, but proline-rich domains are required

Cell migration requires the constant modification of cellular shape by reorganization of the actin cytoskeleton. The pentameric Scar/WAVE regulatory complex (WRC) is the main catalyst of pseudopod and lamellipodium formation. Its actin nucleation activity has been attributed to its ability to combine monomeric actin and Arp2/3 complex through the VCA domain of Scar/WAVE, while other regions of the complex are typically thought to mediate spatial and temporal regulation and have no direct role in actin polymerization. Here we show that the Scar/WAVE with its VCA domain deleted can still induce the formation of morphologically normal actin protrusions. Equivalent results are seen in B16-F1 mouse melanoma cells and Dictyostelium discoideum cells. This actin polymerization occurs independently of the Arp2/3 complex, whose recruitment to the leading edge is greatly reduced by the loss of the VCA domain. We also expressed Scar/WAVE with VCA and polyproline domains both deleted. In Dictyostelium cells, these were only active if WASP (which contains its own proline-rich domain) was available. Similarly, in B16-F1 cells both Abi and WAVE proline-rich domains needed to be deleted before the function of the WRC was lost. Thus we conclude that proline-rich domains play a central role in actin nucleation. Our data demonstrate a new actin nucleation mechanism of the WRC that is independent of its VCA domain and the Arp2/3 complex. We also show that proline-rich domains are more fundamental than has been thought. Together, these findings suggest a new mechanism for WRC action.

cell biology↗

bioRxiv2018

Akt kinases are required for efficient feeding by macropinocytosis in Dictyostelium

Macropinocytosis is an actin-driven process of large-scale, non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum. In Dictyostelium, macropinocytic cups are organised by macropinocytic patches in the plasma membrane. These contain activated Ras, Rac and PI(3,4,5)P3 and direct actin polymerisation to their periphery. Here, we show that a classical (PkbA) and a variant (PkbR1) Akt protein kinase acting downstream of PI(3,4,5)P3 are together are near-essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Akt targets identified by phosphoproteomics were highly enriched in small G-protein regulators, including the RhoGAP GacG. GacG knockout mutants make few macropinosomes but instead redeploy their cytoskeleton from macropinocytosis to motility, moving rapidly but taking up little fluid. The function of Akt in cell feeding through control of macropinosome size has implications for cancer cell biology.\n\nSummary statementDictyostelium amoebae feed by macropinocytosis in a PIP3 dependent manner. In the absence of PI3-kinases or the downstream Akt protein kinases, cells have smaller macropinosomes and nearly abolished fluid uptake.

cell biology↗

bioRxiv2018

Triparental inheritance in Dictyostelium

Sex promotes the recombination and reassortment of genetic material and is prevalent across eukaryotes. In social amoebae sex involves a promiscuous mixing of cytoplasm before zygotes consume the majority of cells. We report here the first genomewide characterisation of meiotic progeny in Dictyostelium discoideum. We find that recombination occurs at high frequency in pairwise crosses between all three mating types, despite the absence of the SPO11 enzyme that is normally required to initiate crossover formation. In crosses involving three strains, transient fusions involving more than two gametes frequently lead to triparental inheritance, with recombined nuclear haplotypes inherited from two parents and the mitochondrial genome from a third. Cells that do not contribute genetically to the Dictyostelium zygote nucleus thereby have a stake in the next haploid generation. We suggest that this lateral transfer helps to enforce cooperation in this confictual system.

genetics↗