Biology subjects

Pedersen, R. T.

Publications and source records attributed to Pedersen, R. T..

6 recordsLinked to original sources

bioRxiv2025

Reconstitution of lamin assembly on nuclear pore complex-containing membranes

Intermediate filaments called lamins line the metazoan nuclear envelope and organize the nucleus and genome. Unlike actin and microtubules, purified intermediate filament proteins assemble into non-physiological structures, making it difficult to connect lamin functions to their assembly and regulation. To overcome this challenge and shed light on physiological lamin assembly mechanisms, we conducted biochemical studies of lamin-B3 endogenously present in Xenopus laevis egg extracts, which recapitulate physiological context. When we mimicked nucleoplasm conditions, which would support assembly of lamin filaments in in-tact cells, lamin-B3 assembled into higher-order structures resembling filamentous meshworks without accompanying nuclear assembly. This ectopic lamin assembly occurs on nuclear pore complex-containing membranes, but does not apparently recruit known nuclear lamina components, demonstrating that a lamin assembly process is partially separable from the rest of the nuclear lamina and nucleus. This assembly assay in the physiological context of cellular components opens the door to further dissecting nuclear lamina function in nuclear organization. SummaryMimicking nucleoplasm conditions in Xenopus laevis egg extracts triggers a lamin assembly reaction independent of nucleus assembly, giving new insights into potential lamin assembly mechanisms.

cell biology↗

bioRxiv2025

Force-insensitive myosin-I enhances endocytosis robustness through actin network-scale collective ratcheting

Force production by Type-I myosins influences endocytic progression in many cell types. Since different myosin-I isoforms exhibit distinct force-dependent kinetic properties, it is important to investigate how these properties affect endocytic outcomes, and the mechanisms through which myosin-I contributes to endocytosis. To this end, we adapted our agent-based simulations of endocytic actin networks and incorporated nonprocessive, single-headed myosin motors at the base of the endocytic pit. We varied the unbinding rate and the force dependence of myosin unbinding. Our results revealed that the inclusion of myosin motors facilitated endocytic internalization, but only under kinetic regimes with rapid and less force-sensitive unbinding. Conversely, slow or strongly force-dependent unbinding impeded endocytic progression. As membrane tension increased, the boundary between assistive and inhibitory phases shifted, allowing the myosins to assist over larger regions of the kinetic landscape. Myosin-Is contribution to internalization could not be explained by direct force transduction or increased actin assembly. Instead, the myosins collectively bolstered the robustness of internalization by limiting pit retraction. Significance StatementO_LIType-I myosins with varying force sensitivity levels participate in different membrane deformation pathways, but the mechanistic link between molecular biophysical properties and cellular function remains poorly understood. C_LIO_LIThe authors analyze a computational model of endocytosis with type-I myosins and find that myosins with lower force sensitivity assist endocytosis by reducing backsliding along the internalization trajectory, while myosins with higher force sensitivity stall endocytosis by sequestering actin in non-productive orientations. C_LIO_LIThese results introduce a new perspective on the function of type-I myosins in membrane reshaping: as a collective emergent property rather than the sum of individual force-generating motors. C_LI

biophysics↗

bioRxiv2023

BuGZ exhibits guanine nucleotide exchange factor toward tubulin

- and {beta}-tubulin form heterodimers, with GTPase activity, that assemble into microtubules. Like other GTPases, the nucleotide-bound state of tubulin heterodimers controls whether the molecules are in a biologically active or inactive state. While -tubulin in the heterodimer is constitutively bound to GTP, {beta}-tubulin can be bound to either GDP (GDP-tubulin) or GTP (GTP-tubulin). GTP-tubulin hydrolyzes its GTP to GDP following assembly into a microtubule and, upon disassembly, must exchange its bound GDP for GTP to participate in subsequent microtubule polymerization. Tubulin dimers have been shown to exhibit rapid intrinsic nucleotide exchange in vitro, leading to a commonly accepted belief that a tubulin guanine nucleotide exchange factor (GEF) may be unnecessary in cells. Here, we use quantitative binding assays to show that BuGZ, a spindle assembly factor, binds tightly to GDP-tubulin, less tightly to GTP-tubulin, and weakly to microtubules. We further show that BuGZ promotes the incorporation of GTP into tubulin using a nucleotide exchange assay. The discovery of a tubulin GEF suggests a mechanism that may aid rapid microtubule assembly dynamics in cells.

biochemistry↗

bioRxiv2023

An endocytic myosin essential for plasma membrane invagination powers motility against resistance

Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse mechanochemical activities, ranging from powerful contractility against mechanical loads to force-sensitive anchoring. To better understand the essential molecular contribution of myosin to endocytosis, we studied the in vitro force-dependent kinetics of the Saccharomyces cerevisiae endocytic type I myosin called Myo5, a motor whose role in clathrin-mediated endocytosis has been meticulously studied in vivo. We report that Myo5 is a low-duty-ratio motor that is activated [~]10-fold by phosphorylation, and that its working stroke and actin-detachment kinetics are relatively force-insensitive. Strikingly, the in vitro mechanochemistry of Myo5 is more like that of cardiac myosin than like that of slow anchoring myosin-1s found on endosomal membranes. We therefore propose that Myo5 generates power to augment actin assembly-based forces during endocytosis in cells. SummaryPedersen, Snoberger et al. measure the force-sensitivity of the yeast endocytic the myosin-1 called Myo5 and find that it is more likely to generate power than to serve as a force-sensitive anchor in cells. Implications for Myo5s role in clathrin-mediated endocytosis are discussed.

cell biology↗

bioRxiv2021

An organelle inheritance pathway during polarized cell growth

Some organelles cannot be synthesized anew, so they are segregated into daughter cells during cell division. In Saccharomyces cerevisiae, daughter cells bud from mother cells and are populated by organelles inherited from the mothers. To determine whether this organelle inheritance occurs in a stereotyped manner, we tracked organelles using fluorescence microscopy. We describe a program for organelle inheritance in budding yeast. The cortical endoplasmic reticulum (ER) and peroxisomes are inherited concomitant with bud emergence. Next, vacuoles are inherited in small buds, followed closely by mitochondria. Finally, the nucleus and perinuclear ER are inherited when buds have nearly reached their maximal size. Because organelle inheritance timing correlates with bud morphology, which is coupled to the cell cycle, we tested whether organelle inheritance order is controlled by the cell cycle. By arresting cell cycle progression but allowing continued bud growth, we determined that organelle inheritance still occurs without cell cycle progression past S-phase, and that the general inheritance order is maintained. Thus, organelle inheritance follows a preferred order during polarized cell division, but it is not controlled exclusively by cell cycle signaling. Summary statementOrganelles are interconnected by contact sites, but they must be inherited from mother cells into buds during budding yeast mitosis. We report that this process occurs in a preferred sequence.

cell biology↗

bioRxiv2019

Cargo licenses maturation of stalled endocytic sites through a regulatory checkpoint

During clathrin-mediated endocytosis (CME), over 50 different proteins assemble on the plasma membrane to reshape it into a cargo-laden vesicle. It has long been assumed that cargo triggers local CME site assembly in Saccharomyces cerevisiae based on the discovery that cortical actin patches clustered near exocytic sites are CME sites. Quantitative imaging data reported here lead to a radically different view of which CME steps are regulated and which steps are deterministic. We quantitatively and spatially describe progression through the CME pathway and pinpoint a cargo-sensitive regulatory transition point that governs progression from the initiation phase of CME to the internalization phase. Thus, site maturation, rather than site initiation, accounts for the previously observed polarized distribution of actin patches in this organism. While previous studies suggested that cargo ensures its own internalization by regulating either CME initiation rates or frequency of abortive events, our data instead identify maturation through a checkpoint in the pathway as the cargo-sensitive step. SummaryPedersen, Hassinger, et al. investigate steps of the clathrin-mediated endocytosis pathway that are subject to regulation. They report position-dependent differences in endocytic site maturation rates in polarized cells and suggest that cargo controls endocytic internalization through tuning site maturation rather than site initiation.

cell biology↗