Biology subjects

Titus, M. A.

Publications and source records attributed to Titus, M. A..

7 recordsLinked to original sources

bioRxiv2025

Filopodia numbers impact chemotactic migration speed

Migrating cells sense and respond to external chemical and physical cues, enabling them to efficiently reach their destinations. Filopodia are slender actin-filled membrane protrusions implicated in interacting with the extracellular environment in many contexts, such as neuronal growth cone guidance and the capture of prey by immune cells and unicellular organisms. The role of filopodia in chemotactic guidance in fast-moving amoeboid cells has not been well-studied. The social amoeba Dictyostelium relies on chemotaxis for development and finding food, making it an excellent system for investigating the role of filopodia in amoeboid chemotaxis. Stimulation of amoebae with the chemoattractant cAMP activates a transient increase of filopodia formation by recruiting the filopodial myosin DdMyo7 to the cell cortex. Filopodia formation is biased towards the source of chemoattractant, yet myo7 null cells that lack filopodia exhibit normal directional migration. However, cells either lacking filopodia or having increased numbers of filopodia move more slowly than those with wildtype numbers of filopodia. Thus, while filopodia are dispensable for detection of chemical gradients by amoeboid cells, changes in filopodia number can impact their migration speed possibly due to altering cell-substrate adhesion. In briefFilopodia formation in chemotactic amoeboid cells is stimulated by chemoattractant and biased towards the gradient source. Lack of filopodia does not impair directed migration but rather reduces cell speed. Changes in filopodia number correlate with the speed of chemotactic cells suggesting a role for these extension in tuning adhesion for optimal migration. HighlightsO_LIFilopodia formation in chemotactic amoeboid cells is biased towards the source of the gradient. C_LIO_LIChemotactic Dictyostelium amoebae lacking filopodia migrate with directional persistence. C_LIO_LIMigration speed is altered by filopodia number. C_LI

cell biology↗

bioRxiv2025

Modulation of Filopodial Myosin Function

MyTH-FERM (MF) myosins are essential for filopodia formation in both Metazoa and Amoebozoa (Dictyostelium DdMyo7; mammalian Myo10) but their roles in filopodia formation are not fully understood. Taking advantage of a mutation in the highly conserved actin binding interface of another MF myosin, the jordan (jd) mutation of Myo15A that reduces its function, the impact of altering the myosin-F-actin interaction on filopodia formation was investigated. The jd mutation, a D to G substitution (Gong et al, 2022, Sci Adv), was introduced into DdMyo7 or Myo10 and filopodia formation assessed by quantitative analysis of number, length and myosin tip enrichment. The mutation significantly decreased filopodia initiation and tip intensity of both DdMyo7 and Myo10. This is consistent with impairment of myosin-based reorganization of cortical actin at the cortex, with myosins in the initiation complex remaining at the tip as the filopodium extends. The DdMyo7 jd mutation did not affect filopodia length. In the case of Myo10, while the jd mutation reduced Myo10 velocity of intrafilopodial motility by 40%, resulting in reduced levels of myosin at the filopodia tip, the jd mutant did not consistently affect the length of filopodia in different cell lines. This indicates that the major role of Myo10 may not be to promote filopodia elongation by reducing membrane tension, as has been proposed. Rather, the phenotype of the DdMyo7 and Myo10 jd mutants indicates that the major role of evolutionarily diverse filopodial myosins is to re-organize actin filaments at the membrane-cortex interface during the critical initiation step.

cell biology↗

bioRxiv2025

Myosin 2 drives actin contractility in fast-crawling species outside of the amorphean lineage

Myosin 2-dependent actin contractility drives essential cell functions including fast crawling motility in animal cells, Dictyostelium amoebae, and other species from the Amorphea lineage. Whether and how species outside this single eukaryotic group can generate contractile actin networks has been largely unexplored. We demonstrate that Naegleria, an amoeba from the Heterolobosea--an evolutionarily distant eukaryotic lineage that includes the fastest known crawling eukaryotes--expresses three distinct Myosin 2 homologs. Using biochemical assays and immunofluorescence, we show that these Myosin 2 proteins bind cellular actin networks and that these networks generate ATP-dependent contractility. By identifying additional Myosin 2 homologs in dozens of additional heterolobosean amoebae (but not obligate flagellates), we find a widespread correlation within this group between crawling behavior and contractile actin networks. This correlation includes the amoeba Vahlkampfia avara, which we demonstrate can crawl at speeds exceeding 180 m/min and has contractile actin networks and Myosin 2 homologs. These findings show that Myosin 2-driven contractility exists beyond Amorphea and is associated with diverse, fast-crawling cell types. Expanding the taxonomic breadth of actin network contractility impacts our basic understanding of cell motility, evolutionary biology, and of the fundamental biology of human pathogens that rely on fast cell migration.

cell biology↗

bioRxiv2024

Oxidative Phosphorylation Inhibition in Different Prostate Cancer Models and the Interplay with Androgen Receptor Signaling

IntroductionIn prostate cancer (PCa), androgen receptor signaling stimulates both glycolysis and oxidative phosphorylation (OxPhos). Early-stage prostate cancer is particularly reliant on OxPhos for its bioenergetic needs. OxPhos inhibitors have entered clinical trials. Here we investigated their interplay with androgens in different PCa cell lines. MethodsWe investigated the effects on PCa cell viability of an ATPase inhibitor (oligomycin) and a complex 1 inhibitor (IACS-010759) in the presence or absence of low testosterone concentrations in vitro. Both androgen-sensitive and insensitive PCa cell lines were used. The effects were assessed using MTT assay, flow cytometry and cell morphology. ResultsTreatment with oligomycin resulted in massive apoptotic death of VCAP cells in castrate conditions within 48 hours, but the simultaneous addition of low testosterone levels restored VCAP cell viability. However, complex 1 inhibition with IACS-010759 increased cell viability, which was further promoted in the presence of testosterone. Both oligomycin and IACS-010759 dramatically decreased viability in LNCaP cells, while testosterone had a small but statistically non-significant effect. The antitumor effect of OxPhos inhibitors was smaller in LNCaP-C4-2B compared to LNCaP cells. OxPhos inhibitors slightly decreased proliferation rates in androgen-independent PC3 cells and HEK293 cells. Oligomycin on LNCaP-C4-2B and PC3 cells resulted in an increased number of cells in G0-G1 phase and decreased in S-phase and G2M phase, rather than massive apoptosis. ConclusionThere is an interplay between androgen signaling and OxPhos in androgen-dependent PCa cells. Complex 1 inhibitors should be used with caution, given potential pro-tumorigenic effects in subsets of PCa cells.

cancer biology↗

bioRxiv2024

Reduced PaxillinB localization to cell-substrate adhesions promotes cell migration in Dictyostelium

Many cells adhere to the extracellular matrix for efficient cell migration. This adhesion is mediated by focal adhesions, a protein complex linking the extracellular matrix to the intracellular cytoskeleton. Focal adhesions have been studied extensively in Metazoan mesenchymal cells, but recent research in physiological contexts and amoeboid cells suggest that focal adhesion regulation differs from the mesenchymal focal adhesion paradigm. While focal adhesion machinery predates the origin of Metazoans, focal adhesion formation and regulation during non-Metazoan cell migration is largely unexplored. We used Dictyostelium discoideum to investigate novel mechanisms and the evolution of focal adhesion regulation, as Dictyostelium are non-Metazoans that form cell-substrate adhesion structures for migration. We show that PaxillinB, the Dictyostelium homologue of Paxillin, localizes to dynamic cell-substrate adhesions. As expected, PaxillinB mutations decreased the number of cell-substrate adhesions. Unexpectedly, however, decreased cell-substrate adhesion number led to an increase in cell migration speed. These findings are in direct contrast to Paxillin function at focal adhesions and regulation of cell migration in mammalian cells, challenging the established focal adhesion model and providing insight into the evolution of cell-substrate adhesions and Paxillin function during cell migration. Significance StatementO_LIFocal adhesions are understudied in non-mesenchymal, non-Metazoan systems. C_LIO_LIThe authors characterize PaxillinB, the Dictyostelium homologue of Paxillin, during Dictyostelium cell migration. Reducing cell-substrate adhesion number via PaxillinB mutations lead to decreased cell adhesion and, surprisingly, increased cell migration speeds. C_LIO_LIThese findings suggest Dictyostelium cells form cell-substrate adhesions that act as molecular "brakes" during cell migration and provides unique insight into the evolution of Paxillin as a regulator of cell-substrate adhesion function and formation during cell migration. C_LI

cell biology↗

bioRxiv2021

VASP mediated actin dynamics activate and recruit a filopodia myosin

Filopodia are thin, actin-based structures that cells use to interact with their environments. Filopodia initiation requires a suite of conserved proteins but the mechanism remains poorly understood. The actin polymerase VASP and a MyTH-FERM (MF) myosin, DdMyo7 in amoeba, are essential for filopodia initiation. DdMyo7 is localized to dynamic regions of the actin-rich cortex. Analysis of VASP mutants and treatment of cells with anti-actin drugs shows that myosin recruitment and activation in Dictyostelium requires localized VASP-dependent actin polymerization. Targeting of DdMyo7 to the cortex alone is not sufficient for filopodia initiation; VASP activity is also required. The actin regulator locally produces a cortical actin network that activates myosin and together they shape the actin network to promote extension of parallel bundles of actin during filopodia formation. This work reveals how filopodia initiation requires close collaboration between an actin binding protein, the state of the actin cytoskeleton and MF myosin activity.

cell biology↗

bioRxiv2020

The actin networks of chytrid fungi reveal evolutionary loss of cytoskeletal complexity in the fungal kingdom

Cells from across the eukaryotic tree use actin polymers and a number of conserved regulators for a wide variety of functions including endocytosis, cytokinesis, and cell migration. Despite this conservation, the actin cytoskeleton has undergone significant evolution and diversification, highlighted by the differences in the actin cytoskeletal networks of mammalian cells and yeast. Chytrid fungi diverged before the emergence of the Dikarya (multicellular fungi and yeast), and therefore provide a unique opportunity to study the evolution of the actin cytoskeleton. Chytrids have two life stages: zoospore cells that can swim with a flagellum, and sessile sporangial cells that, like multicellular fungi, are encased in a chitinous cell wall. Here we show that zoospores of the amphibian-killing chytrid Batrachochytrium dendrobatidis (Bd) build dynamic actin structures that resemble those of animal cells, including pseudopods, an actin cortex, and filopodia-like actin spikes. In contrast, Bd sporangia assemble actin patches similar to those of yeast, as well as perinuclear actin shells. Our identification of actin cytoskeletal elements in the genomes of five species of chytrid fungi indicate that these actin structures are controlled by both fungal-specific components as well as actin regulators and myosin motors found in animals but not other fungal lineages. The use of specific small molecule inhibitors indicate that nearly all of Bds actin structures are dynamic and use distinct nucleators: while pseudopods and actin patches are Arp2/3-dependent, the actin cortex appears formin-dependent, and actin spikes require both nucleators. The presence of animal- and yeast-like actin cytoskeletal components in the genome combined with the intermediate actin phenotypes in Bd suggests that the simplicity of the yeast cytoskeleton may be due to evolutionary loss.

cell biology↗