Session K07: Intracellular Transport II: Transport at the Cellular Scale

Transport and Maturation of Interacting Organelles in Axons

Many organelles are transported long distances through the long narrow projections of neurons. The organelles pass by each other, and in some cases fuse permanently or transiently, exchanging internal material. We explore how the distribution of organelles and their contents depends on transport and interaction parameters, particularly in the context of long linear or branched cell geometries.

First, we will discuss the maintenance of axonal mitochondria stationed at distal regions of high metabolic demand. The turnover of mitochondrial content requires replenishment by delivery of new material from the soma, either through exchanging whole organelles between a stationary and motile population or through transient fusion and fission of motile mitochondria with permanently stationary ones. We show that both mechanisms are equivalent in the mean-field sense, with optimal mitochondrial health requiring intermediate fractions of the population in the motile state, and only a few exchange events per every round-trip of a motile mitochondrion. We also use agent-based simulations to show how transient fusion leads to more robust mitochondrial health, particularly in response to autophagic mechanisms for selectively recycling unhealthy mitochondria.

Additionally, we explore the maturation dynamics of neuronal autophagosomes, which are produced in the distal tip of the axon, and fuse with degradatively active endolysosomes en route to the soma. Parameterizing our model against experimental data, we demonstrate that individual autophagosomes must fuse with only a few endolysosomes, primarily in the distal region, enabling relatively uniform distributions of organelles throughout the axon. The mathematical models presented here provide a quantitative picture of how transport behaviors, fusion, and the geometry of neuronal projections couple together to determine the distribution and functional state of organelles in mammalian axons.   

Trafficking Mechanics of Inter-Synaptic Vesicle Exchange Support in Synaptic Transmission

Mature presynapses share proteins with neighboring presynapses via intracellular axonal transport. These proteins are predominantly shared using neurotransmitter carrying synaptic vesicles, through a process called Inter-Synaptic-Vesicle-Exchange (ISVE). Further, ISVE may also support synaptic transmission by providing a pool of vesicles during activity. While the mechanics of axonal synaptic vesicle transport have been studied previously, the capture of vesicles by neighboring presynapses dominate the efficiency of ISVE and the capture process is less understood. In this presentation we will discuss evidence that the structure of the actin presynaptic cytoskeleton, through the ARP2/3 branching complex, is important for vesicle capture. We utilize high-resolution single vesicle fluorescence microscopy in live cells to measure ISVE transport at the nanometer and millisecond scales. We show that acutely inhibiting the ARP2/3 complex results in reduced vesicle capture and synaptic transmission. We also show that acute loss of the actin-based transport motor myosin-V reduces vesicle capture. Lastly, we show how actin structure and myosin-V function mediate activity-dependent capture to support synaptic transmission.   

Distinguishing Neurotransmitter Carrying Pause Mechanics During Axonal Trafficking

Mature presynapses share neurotransmitter carrying vesicles with neighboring presynapses using a process called Inter-Synaptic Vesicle Exchange (ISVE). ISVE has been considered as a pathway to support synaptic transmission, by providing an extra pool of vesicles during activity. Understanding the mechanics of ISVE would thus allow us to better model the efficiency with which ISVE supports synaptic transmission. However, there are major gaps in our understanding about how ISVE vesicles transport along the axon in order to support transmission. ISVE vesicles utilize molecular motor transport, which is fast and efficient, but are predominantly rate-limited by vesicle pausing along the axon. We utilize high-resolution single vesicle fluorescence microscopy in live cells to measure ISVE transport at the nanometer and millisecond scales. We distinguish vesicles undergoing axonal transport, and use a computational approach to characterize motor-driven motion from pausing. We further developed a computational approach to quantify mechanics of vesicle pauses along the axonal cytoskeleton. We show that vesicles likely utilize multi-motor coordination in order to navigate the complex axonal cytoskeleton. We show that loss of actin-based transport results in altered pausing mechanics.   

Modeling intra-cellular insulin transport dynamics in pancreatic Beta cells

In this talk, I will discuss the role of cytoskeletal-mediated transport (by microtubules) in regulating insulin dynamics in pancreatic cells. Due to the increasing prevalence of diabetes and related disorders, understanding how individual cells regulate insulin availability and secretion in response to glucose stimulation is of utmost importance. While it has been known for decades that dysregulated microtubule dynamics alter insulin secretion, their role in insulin regulation has been murky. Here I use computational modeling to demonstrate a new mechanism by which apparently random trafficking of insulin on a random network of microtubules regulates the intra-cellular localization and availability of insulin. These results demonstrate that microtubule mediated trafficking negatively regulates insulin secretion. Accompanying experiments confirm this hypothesis and demonstrate the potential for targeting of microtubule dynamics to provide a new avenue to manipulate insulin secretion.   

Intracellular cargo transport time statistics that result from competing transport mechanisms.

We consider intracellular transport of cargo when molecular motor-driven transport along microtubules is directed towards the target in the interior of the cell.  Diffusive motion of cargo - when the motors disengage from microtubules - allows the transport in the opposite direction, with the possibility of reaching the cell membrane.  We find that when the switching between the two transport mechanisms is rapid, the asymmetric placement of the target gives the cell a sensitive control of the mean first passage time (MFPT) for reaching the membrane as a function of the location of the source of cargo and of the asymmetry.  At lower rates of switching, we find a transition to much lower MFPTs due to the possibility of a purely diffusive escape.  Here again, the degree of asymmetry of the target placement can lead to rich phenomenology.  We map out the phase diagram for different regimes in the space of parameters and discuss other statistical quantities - such as properties of density profiles that are established due to the competition between the two transport mechanisms.     

Optimal cytoskeletal filament dynamics for intracellular transport

The transport of cargo within cells is typically caried out by a combination of active motor-driven motion on cytoskeletal filaments and diffusion in the cytoplasm. It is known that the  morpholgy of the cytoskeletal network differs between cell types and plays a significant role in determining transport. However, the network is not static but can turnover on the same time scale as the transport of cargo on the network. Here, we study the transport of cargo carried by myosin motors on a dynamic actin network. We use a stochastic simulation model that accounts for both active transport along filaments as well as passive diffusion in the cytoplasm and incorporates the dynamics of the explicitly represented actin network. We show how filament treadmilling rates affect cargo transport along with filament lengths, densities and motor attachment/detachment rates. In particular, using simulations and simple analytics, we show that the turnover rates of the network can be optimized for fast transport and that the optimal dynamics regime is consistent with in vivo conditions. Additionally the optimal regime can be tuned by both cargo properties and filament densities and lengths suggesting new ways for cells to regulate transport.   

A Slowing of Neurofilament Transport and an Increase in Neurofilament Influx Both Contribute to the Radial Growth of Axons During Development

Neurofilaments (NFs) are abundant space-filling cytoskeletal protein polymers in axons. They are assembled in the nerve cell body and transported towards the nerve terminal along microtubule tracks at an average velocity of 0.1 to 1mm/day. During postnatal development, NFs accumulate in axons causing them to expand radially. In this way, NFs determine one of the basic cable properties of axons that influences axonal conduction velocity.  Theoretically, this NF accumulation can occur by an increased expression and influx of NFs from the cell body and/or by a slowing of NF transport in the axon.  To explore the relative contribution of these two mechanisms, we developed a computational model that simulates NF transport and axon growth. We compared the model to published experimental data on the kinetics of NF transport obtained by radio-isotopic pulse-labeling and published experimental data relating axon caliber to NF and microtubule content during axonal growth.  We constrained the model to match the increase in axon caliber and the speed and shape of the distribution of radiolabeled NFs as they traverse the axon during maturation of axons in rat sciatic nerve.  We found that slowing alone is not sufficient to explain the observed NF accumulation and that both a slowing of NF transport and an increase in NF influx from the cell body contribute to the expansion of axons during postnatal development.   

Molecular Quakes and Neurofilament movement

Neurofilaments (NFs) are abundant protein polymers of the axonal cytoskeleton that determine axon caliber, which is important for neuronal function. The NFs are aligned in parallel along the long axis of the axon, spaced apart by a dense border of lateral sidearm projections that interact with neighboring NFs through weak electrostatic forces.  NFs are also cargoes of axonal transport, propelled along microtubule tracks by molecular motors.  These polymer cargos are assembled in the cell body and move along the axon in a stop-and-go manner, alternating between brief bursts of rapid movement interrupted by prolonged pauses. The mechanism for the intermittent nature of this movement is unknown.  Here we show that this can be explained by the stochastic and reversible association of NF sidearms with neighboring NFs. When a driving force is applied to a pausing filament, tension builds along the length of the NF. As sidearm linkages break under this force, the force is born by successively fewer sidearms, resulting in an increased rate of detachment, abrupt release, and punctuated sudden NF movement.  This resembles the movement of tectonic plates along fault lines, where tension buildup is released in punctuated events, the earthquakes. Just like earthquakes, molecular quakes are power-law distributed in amplitude. We demonstrate the model's viability by comparing the predicted behavior with high-resolution kymographic recordings of NF movement in axons.   

Mitochondrial distribution in neurons: Interplay of transport and morphology

Neurons consist of a small cell body (soma) and several extended projections (axons and dendrites). To maintain neuronal health, organelles and other components derived from the soma must be optimally distributed through this extensive morphology. Mitochondria navigate the tree-like branched geometry of neuronal dendrites through bidirectional active transport and regulated stopping. In dendritic trees of the Drosophila visual system, mitochondrial densities increase with distance from the soma. Despite spatial heterogeneity in mitochondrial density and subtree morphology, sister subtrees have balanced total mitochondrial densities. Using mathematical modeling, we show that specific scaling laws for dendritic branching patterns enable robust self-organization of the mitochondrial population for a range of transport behaviors, to maintain equitable distribution across subtrees while still allowing for increased densities at distal tips. Our theoretical predictions are validated by experimental measurements of dendritic structure and mitochondrial transport, demonstrating that the scaling laws are observed in the dendritic trees of the Drosophila visual system.   

Image-based model of diffusion on the endoplasmic reticulum

We use a coarse-grained model of random walks on networks to efficiently simulate the diffusion of biomolecules along the endoplasmic reticulum (ER). Along each ER tubule, the diffusion process may be conceptualized as a one-dimensional gambler’s ruin problem. By computing the mean first passage times (MFPT) to diffuse across the ER network, we connect network structure to the typical time required for synthesized proteins and other biomolecules to traverse the ER. We explore how network dynamics affect the MFPT and apply the model to data from Drosophila melanogaster neuromuscular junctions.   

Protein dispersion and facilitated diffusion in the endoplasmic reticulum

The endoplasmic reticulum (ER) forms a dynamic network of lipid membrane sheets and tubules from the cell nucleus to the periphery. Its many functional roles include the distribution of lipids, ions and proteins throughout the cell, but the impact of morphology on transport remains poorly understood. Pairing our novel propagator-driven approach for fast agent-based simulations of particles diffusing on tubular networks with in vivo data on the spreading of photoactivated membrane proteins, we quantify how local ER network structure determines protein spreading. Our results demonstrate that network structure and diffusion explain much, but not all, of the behavior of ER membrane proteins. Tubule growth and rearrangement are also implicated in determining protein dispersion. We describe a new model for cargo loading and sorting at ER exit sites, relying on `facilitated diffusion’ of secretory cargo to a region of weak binding surrounding a narrow exit site neck. We link local network structure to the rate of cargo accumulation, and compare these results to in vivo measurements following synchronized cargo release. Using theory and live-cell imaging, we highlight the structure-function relationship of the ER as an intracellular transport hub with a uniquely complex morphology.   

Long lived protein demixing in flagella

Cells create different internal compartments by diffusional uncoupling of one part of the cells from another. This helps cells to maintain different protein concentrations in different parts of the cell, which is used for creating compartments with specialized functions. Well known examples are provided by membrane bound organelles such as the nucleus and mitochondria, or phase separated structures such as the nucleolus. In this talk I will discuss another mechanism for creating a diffusionally decoupled region within the cell, which is achieved at the tips of flagella by motor-driven transport of molecules toward the tip. An example of this is provide by the localization of kinesin-13 proteins to the tips of flagella in Giardia [1]. In this talk, I describe a simple physical model of combined directed transport and diffusion that leads to diffusional uncoupling of the flagellar tip from the cell body over time scales that are exponential in the length of the flagellum. I comment on the theoretical possibility of this mechanism providing the graded staircase geometry of stereocilia in hair cells of the inner ear.

 

References:

[1]   S. G. McInally, J. Kondev, and S. C. Dawson, “Length-dependent disassembly maintains four different flagellar lengths in Giardia,” eLife, vol. 8, p. e48694, Dec. 2019, doi: 10.7554/eLife.48694.   

Large deformations in the cytoplasm and cytoskeleton plasticity

Cells spread, proliferate, migrate, divide, and die. Large deformations of the cytoplasm are associated with these processes.  Considerable research has generated a mechanical description of the cytoplasm; however, most of this work has concentrated on the small deformation linear regime. In this talk, I will describe the physics of large cytoplasm deformations.

In this experiment work, we use an electrical magnetic tweezer to pull ferromagnetic particles through the cytoplasm. We induce large deformations and measure the plasticity of the cytoplasm.  Further, we visualize how fluorescently-tagged cytoskeleton filaments: microtubules and vimentin reorganize themselves during this process. We find that the strain rate of large deformations has a distinct limit, independent of the applied force. This strain rate limit is dictated by the cytoskeleton dynamics. It can be altered by chemical treatments that either change the cytoskeleton density or inhibit the cytoskeleton dynamics. 

We were also able to differentiate the role of each filament type. In the mouse embryonic fibroblast cell line, we find the microtubules and the intermediate filaments are important in the cytoplasm plastic deformation due to their abundance. In addition, they act synergistically to generate deformation resistance: removal of either filament completely changes the physics of the large deformation.

This research joins two longtime biophysics concepts together: the capacity of the cell to deform over time,e.g. cargo transport, cell migration, cell division, and the cytoskeleton dynamics. We hope this study can enrich the mechanical and physical understanding of the complicated and fascinating cellular biosystem.