Colony context and size-dependent compensation mechanisms give rise to variations in nuclear growth trajectories.

To investigate the fundamental question of how cellular variations arise across spatiotemporal scales in a population of identical healthy cells, we focused on nuclear growth in hiPS cell colonies as a model system. We generated a 3D timelapse dataset of thousands of nuclei over multiple days, and developed open-source tools for image and data analysis and an interactive timelapse viewer for exploring quantitative features of nuclear size and shape. We performed a data-driven analysis of nuclear growth variations across timescales. We found that individual nuclear volume growth trajectories arise from short timescale variations attributable to their spatiotemporal context within the colony. We identified a strikingly time-invariant volume compensation relationship between nuclear growth duration and starting volume across the population. Notably, we discovered that inheritance plays a crucial role in determining these two key nuclear growth features while other growth features are determined by their spatiotemporal context and are not inherited.

Spatiotemporal analysis of axonal autophagosome-lysosome dynamics reveals limited fusion events and slow maturation.

Macroautophagy is a homeostatic process required to clear cellular waste. Neuronal autophagosomes form constitutively in the distal tip of the axon and are actively transported toward the soma, with cargo degradation initiated en route. Cargo turnover requires autophagosomes to fuse with lysosomes to acquire degradative enzymes; however, directly imaging these fusion events in the axon is impractical. Here we use a quantitative model, parameterized and validated using data from primary hippocampal neurons, to explore the autophagosome maturation process. We demonstrate that retrograde autophagosome motility is independent of fusion and that most autophagosomes fuse with only a few lysosomes during axonal transport. Our results indicate that breakdown of the inner autophagosomal membrane is much slower in neurons than in nonneuronal cell types, highlighting the importance of this late maturation step. Together, rigorous quantitative measurements and mathematical modeling elucidate the dynamics of autophagosome-lysosome interaction and autophagosomal maturation in the axon.

Optimizing microtubule arrangements for rapid cargo capture.

Cellular functions such as autophagy, cell signaling, and vesicular trafficking involve the retrograde transport of motor-driven cargo along microtubules. Typically, newly formed cargo engages in slow undirected movement from its point of origin before attaching to a microtubule. In some cell types, cargo destined for delivery to the perinuclear region relies on capture at dynein-enriched loading zones located near microtubule plus ends. Such systems include extended cell regions of neurites and fungal hyphae, where the efficiency of the initial diffusive loading process depends on the axial distribution of microtubule plus ends relative to the initial cargo position. We use analytic mean first-passage time calculations and numerical simulations to model diffusive capture processes in tubular cells, exploring how the spatial arrangement of microtubule plus ends affects the efficiency of retrograde cargo transport. Our model delineates the key features of optimal microtubule arrangements that minimize mean cargo capture times. Namely, we show that configurations with a single microtubule plus end abutting the distal tip and broadly distributed other plus ends allow for efficient capture in a variety of different scenarios for retrograde transport. Live-cell imaging of microtubule plus ends in Aspergillus nidulans hyphae indicates that their distributions exhibit these optimal qualitative features. Our results highlight important coupling effects between the distribution of microtubule tips and retrograde cargo transport, providing guiding principles for the spatial arrangement of microtubules within tubular cell regions.

Diffusive search and trajectories on tubular networks: a propagator approach.

Several organelles in eukaryotic cells, including mitochondria and the endoplasmic reticulum, form interconnected tubule networks extending throughout the cell. These tubular networks host many biochemical pathways that rely on proteins diffusively searching through the network to encounter binding partners or localized target regions. Predicting the behavior of such pathways requires a quantitative understanding of how confinement to a reticulated structure modulates reaction kinetics. In this work, we develop both exact analytical methods to compute mean first passage times and efficient kinetic Monte Carlo algorithms to simulate trajectories of particles diffusing in a tubular network. Our approach leverages exact propagator functions for the distribution of transition times between network nodes and allows large simulation time steps determined by the network structure. The methodology is applied to both synthetic planar networks and organelle network structures, demonstrating key general features such as the heterogeneity of search times in different network regions and the functional advantage of broadly distributing target sites throughout the network. The proposed algorithms pave the way for future exploration of the interrelationship between tubular network structure and biomolecular reaction kinetics.

Hitching a Ride: Mechanics of Transport Initiation through Linker-Mediated Hitchhiking.

In contrast to the canonical picture of transport by direct attachment to motor proteins, recent evidence shows that a number of intracellular "cargos" navigate the cytoplasm by hitchhiking on motor-driven "carrier" organelles. We describe a quantitative model of intracellular cargo transport via hitchhiking, examining the efficiency of hitchhiking initiation as a function of geometric and mechanical parameters. We focus specifically on the parameter regime relevant to the hitchhiking motion of peroxisome organelles in fungal hyphae. Our work predicts the dependence of transport initiation rates on the distribution of cytoskeletal tracks and carrier organelles, as well as the number, length, and flexibility of the linker proteins that mediate contact between the carrier and the hitchhiking cargo. Furthermore, we demonstrate that attaching organelles to microtubules can result in a substantial enhancement of the hitchhiking initiation rate in tubular geometries such as those found in fungal hyphae. This enhancement is expected to increase the overall transport rate of hitchhiking organelles and lead to greater efficiency in organelle dispersion. Our results leverage a quantitative physical model to highlight the importance of organelle encounter dynamics in noncanonical intracellular transport.

Multimodal transport and dispersion of organelles in narrow tubular cells.

Intracellular components explore the cytoplasm via active motor-driven transport in conjunction with passive diffusion. We model the motion of organelles in narrow tubular cells using analytical techniques and numerical simulations to study the efficiency of different transport modes in achieving various cellular objectives. Our model describes length and time scales over which each transport mode dominates organelle motion, along with various metrics to quantify exploration of intracellular space. For organelles that search for a specific target, we obtain the average capture time for given transport parameters and show that diffusion and active motion contribute to target capture in the biologically relevant regime. Because many organelles have been found to tether to microtubules when not engaged in active motion, we study the interplay between immobilization due to tethering and increased probability of active transport. We derive parameter-dependent conditions under which tethering enhances long-range transport and improves the target capture time. These results shed light on the optimization of intracellular transport machinery and provide experimentally testable predictions for the effects of transport regulation mechanisms such as tethering.