Intracellular Transport Dynamics Research Articles

Interplay Between Intracellular Transport Dynamics and Liquid‒Liquid Phase Separation.

Liquid‒liquid phase separation (LLPS) is a ubiquitous process in which proteins, RNA, and biomolecules assemble into membrane-less compartments, playing important roles in many biological functions and diseases. The current knowledge on the biophysical and biochemical principles of LLPS is largely from in vitro studies; however, the physiological environment in living cells is complex and not at equilibrium. The characteristics of intracellular dynamics and their roles in physiological LLPS remain to be resolved. Here, by using single-particle tracking of quantum dots and dynamic monitoring of the formation of stress granules (SGs) in single cells, the spatiotemporal dynamics of intracellular transport in cells undergoing LLPS are quantified. It is shown that intracellular diffusion and active transport are both reduced. Furthermore, the formation of SG droplets contributes to increased spatial heterogeneity within the cell. More importantly, the study demonstrated that the LLPS of SGs can be regulated by intracellular dynamics in two stages: the reduced intracellular diffusion promotes SG assembly and the microtubule-associated transport facilitates SG coalescences. The work on intracellular dynamics not only improves the understanding of the mechanism of physiology phase separations occurring in nonequilibrium environments but also reveals an interplay between intracellular dynamics and LLPS.

Non-Markovian Persistent Random Walk Model for Intracellular Transport

Transport of vesicles and organelles inside cells consists of constant-speed bidirectional movement along cytoskeletal filaments interspersed by periods of idling. This transport shows many features of anomalous diffusion. In this paper, we develop a non-Markovian persistent random walk model for intracellular transport that incorporates the removal rate of organelles. The model consists of two active states with different speeds and one resting state. The organelle transitions between states with switching rates that depend on the residence time the organelle spends in each state. The mesoscopic master equations that describe the average densities of intracellular transport in each of the three states are the main results of the paper. We also derive ordinary differential equations for the dynamics for the first and second moments of the organelles’ position along the cell. Furthermore, we analyse models with power-law distributed random times, which reveal the prevalence of the Mittag-Leffler resting state and its contribution to subdiffusive and superdiffusive behaviour. Finally, we demonstrate a non-Markovian non-additivity effect when the switching rates and transport characteristics depend on the rate of organelles removal. The analytical calculations are in good agreement with numerical Monte Carlo simulations. Our results shed light on the dynamics of intracellular transport and emphasise the effects of rest times on the persistence of random walks in complex biological systems.

Preferential import of queuosine-modified tRNAs into Trypanosoma brucei mitochondrion is critical for organellar protein synthesis.

Transfer RNAs (tRNAs) are key players in protein synthesis. To be fully active, tRNAs undergo extensive post-transcriptional modifications, including queuosine (Q), a hypermodified 7-deaza-guanosine present in the anticodon of several tRNAs in bacteria and eukarya. Here, molecular and biochemical approaches revealed that in the protozoan parasite Trypanosoma brucei, Q-containing tRNAs have a preference for the U-ending codons for asparagine, aspartate, tyrosine and histidine, analogous to what has been described in other systems. However, since a lack of tRNA genes in T. brucei mitochondria makes it essential to import a complete set from the cytoplasm, we surprisingly found that Q-modified tRNAs are preferentially imported over their unmodified counterparts. In turn, their absence from mitochondria has a pronounced effect on organellar translation and affects function. Although Q modification in T. brucei is globally important for codon selection, it is more so for mitochondrial protein synthesis. These results provide a unique example of the combined regulatory effect of codon usage and wobble modifications on protein synthesis; all driven by tRNA intracellular transport dynamics.

Intracellular transport dynamics revealed by single-particle tracking.

Intracellular transport is the basis for the transfer of matter, energy, and information in cells and is critical to many cellular functions. Within the nonequilibrium environment of living cells, the transport behaviours are far from the traditional motion in liquid but are more complex and active. With the advantage of high spatial and temporal resolution, the single-particle tracking (SPT) method is widely utilized and has achieved great advances in revealing intracellular transport dynamics. This review describes intracellular transport from a physical perspective and classifies it into two modes: diffusive motion and directed motion. The biological functions and physical mechanisms for these two transport modes are introduced. Next, we review the principle of SPT and its advances in two aspects of intracellular transport. Finally, we discuss the prospect of near infrared SPT in exploring the in vivo intracellular transport dynamics.

Intracellular Transport Dynamics of Upconverting Nanoparticles in Living Cells

Intracellular Transport Dynamics of Upconverting Nanoparticles in Living Cells

Quantitative investigation on morphology and intracellular transport dynamics of migrating cells.

In this paper, we present a phase correlation imaging technique employing quantitative phase microscopy to study the migration properties of living cells. The phase correlation map relies on time-lapse quantitative phase stacks, which are achieved by single-shot quantitative phase imaging based on the transport of intensity equation. The phase correlation imaging maps can reveal tiny fluctuations in cellular optical path length and dynamic subdomains in living cells. We find that the morphology dynamics and intracellular mass transport rates are significantly different as cells exhibit different levels of migratory capabilities.

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes.

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the "tri-partite" synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes' normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Membrane mediated motor kinetics in microtubule gliding assays

Motor-based transport mechanisms are critical for a wide range of eukaryotic cell functions, including the transport of vesicle cargos over long distances. Our understanding of the factors that control and regulate motors when bound to a lipid substrate is however incomplete. We used microtubule gliding assays on a lipid bilayer substrate to investigate the role of membrane diffusion in kinesin-1 on/off binding kinetics and thereby transport velocity. Fluorescence imaging experiments demonstrate motor clustering on single microtubules due to membrane diffusion in the absence of ATP, followed by rapid ATP-induced dissociation during gliding. Our experimental data combined with analytical modeling show that the on/off binding kinetics of the motors are impacted by diffusion and, as a consequence, both the effective binding and unbinding rates for motors are much lower than the expected bare rates. Our results suggest that motor diffusion in the membrane can play a significant role in transport by impacting motor kinetics and can therefore function as a regulator of intracellular transport dynamics.

Intracellular transport is accelerated in early apoptotic cells

Intracellular transport of cellular proteins and organelles is critical for establishing and maintaining intracellular organization and cell physiology. Apoptosis is a process of programmed cell death with dramatic changes in cell morphology and organization, during which signaling molecules are transported between different organelles within the cells. However, how the intracellular transport changes in cells undergoing apoptosis remains unknown. Here, we study the dynamics of intracellular transport by using the single-particle tracking method and find that both directed and diffusive motions of endocytic vesicles are accelerated in early apoptotic cells. With careful elimination of other factors involved in the intracellular transport, the reason for the acceleration is attributed to the elevation of adenosine triphosphate (ATP) concentration. More importantly, we show that the accelerated intracellular transport is critical for apoptosis, and apoptosis is delayed when the dynamics of intracellular transport is regulated back to the normal level. Our results demonstrate the important role of transport dynamics in apoptosis and shed light on the apoptosis mechanism from a physical perspective.

Unbinding of Kinesin from Microtubule in the Strongly Bound States Enhances under Assisting Forces.

The ability to predict the cellular dynamics of intracellular transport has enormous potential to impact human health. A key transporter is kinesin-1, an ATP-driven molecular motor that shuttles cellular cargos along microtubules (MTs). The dynamics of kinesins depends critically on their unbinding rate from MT, which varies depending on the force direction applied on the motor, i.e. the force-unbinding rate relation is asymmetric. However, it remains unclear how changing the force direction from resisting (applied against the motion direction) to assisting (applied in the motion direction) alters the kinesin's unbinding and stepping. Here, we propose a theoretical model for the influence of the force direction on the stepping dynamics of a single kinesin. The model shows that the asymmetry of the force-unbinding rate relation is independent of ATP concentration. It also reveals that the synthesis of ATP from backward stepping under assisting forces is less likely than under resisting forces. It then finds that the unbinding of kinesin in the strongly MT-bound kinetic states enhances under assisting forces.

Viral RNA Degradation and Diffusion Act as a Bottleneck for the Influenza A Virus Infection Efficiency

After endocytic uptake, influenza viruses transit early endosomal compartments and eventually reach late endosomes. There, the viral glycoprotein hemagglutinin (HA) triggers fusion between endosomal and viral membrane, a critical step that leads to release of the viral segmented genome destined to reach the cell nucleus. Endosomal maturation is a complex process involving acidification of the endosomal lumen as well as endosome motility along microtubules. While the pH drop is clearly critical for the conformational change and membrane fusion activity of HA, the effect of intracellular transport dynamics on the progress of infection remains largely unclear. In this study, we developed a comprehensive mathematical model accounting for the first steps of influenza virus infection. We calibrated our model with experimental data and challenged its predictions using recombinant viruses with altered pH sensitivity of HA. We identified the time point of virus-endosome fusion and thereby the diffusion distance of the released viral genome to the nucleus as a critical bottleneck for efficient virus infection. Further, we concluded and supported experimentally that the viral RNA is subjected to cytosolic degradation strongly limiting the probability of a successful genome import into the nucleus.

Stochastic dynamics of coupled active particles in an overdamped limit

We introduce a model for Brownian dynamics of coupled active particles in an overdamped limit. Our system consists of several identical active particles and one passive particle. Each active particle is elastically coupled to the passive particle and there is no direct coupling among the active particles. We investigate the dynamics of the system with respect to the number of active particles, viscous friction, and coupling between the active and passive particles. For this purpose, we consider an intracellular transport process as an application of our model and perform a Brownian dynamics simulation using realistic parameters for processive molecular motors such as kinesin-1. We determine an adequate energy conversion function for molecular motors and study the dynamics of intracellular transport by multiple motors. The results show that the average velocity of the coupled system is not affected by the number of active motors and that the stall force increases linearly as the number of motors increases. Our results are consistent with well-known experimental observations. We also examine the effects of coupling between the motors and the cargo, as well as of the spatial distribution of the motors around the cargo. Our model might provide a physical explanation of the cooperation among active motors in the cellular transport processes.

Understanding Non Brownian Dynamics of Intracellular Transport

Understanding insulin granule transport in live beta cells is a complicated task. Traditionally, the diffusion coefficients and the velocity of insulin granules measured via particle tracking techniques are used to characterize the dynamics, which requires the assumption that the dynamics to be either purely diffusive or ballistic. This is not the case for insulin granules. We use a variety of statistical data analysis, to show that insulin granule vesicles in their pathway, which leads to exocytosis performs a subordinated intercellular transport mechanisms, which leads to a statistical anomalous dynamics.

Reversible Photoswitchable DRONPA‐s Monitors Nucleocytoplasmic Transport of an RNA‐Binding Protein in Transgenic Plants

Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state are novel tools for monitoring intracellular protein trafficking. A codon-optimized variant of the reversibly photoswitchable fluorescent protein DRONPA was designed for the use in transgenic Arabidopsis plants. Its codon usage is also well adapted to the mammalian codon usage. The synthetic protein, DRONPA-s, shows photochemical properties and switching behavior comparable to that of the original DRONPA from Pectiniidae both in vitro and in vivo. DRONPA-s fused to the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) under control of the endogenous AtGRP7 promoter localizes to cytoplasm, nucleoplasm and nucleolus of transgenic Arabidopsis plants. To monitor the intracellular transport dynamics of AtGRP7-DRONPA-s, we set up a single-molecule sensitive confocal fluorescence microscope. Fluorescence recovery after selective photoswitching experiments revealed that AtGRP7-DRONPA-s reaches the nucleus by carrier-mediated transport. Furthermore, photoactivation experiments showed that AtGRP7-DRONPA-s is exported from the nucleus. Thus, AtGRP7 is a nucleocytoplasmic shuttling protein. Our results show that the fluorescent marker DRONPA-s is a versatile tool to track protein transport dynamics in stably transformed plants.

Intracellular Transport Dynamics of Endosomes Containing DNA Polyplexes along the Microtubule Network

We have explored the transport of DNA polyplexes enclosed in endosomes within the cellular environment by multiple particle tracking (MPT). The polyplex-loaded endosomes demonstrate enhanced diffusion at short timescales ( t < 7 s) with their mean-square displacement (MSD) 〈Δ x( t) 2 〉 scaling as t 1.25. For longer time intervals they exhibit subdiffusive transport and have an MSD scaling as t 0.7. This crossover from an enhanced diffusion to a subdiffusive regime can be explained by considering the action of motor proteins that actively transport these endosomes along the cellular microtubule network and the thermal bending modes of the microtubule network itself.

Kinetics of prolyl hydroxylation, intracellular transport and C-terminal processing of the tobacco vacuolar chitinase

The dynamics of intracellular transport and processing of one of the vacuolar chitinases of tobacco (Nic-otiana tabacum L.), chitinase A (CHN A; EC 3.2.1.14), was investigated with pulse-chase experiments in conjunction with cell fractionation and immunoprecipitation. Mature CHN A is composed of two domains, the N-terminal cysteine-rich chitin-binding domain and the catalytic domain, linked by a short peptide spacer containing several hydroxyprolines. It is synthetized as a preproprotein with a signal peptide for cotranslational transport into the endoplasmic reticulum (ER) and a C-terminal, vacuolar targeting peptide (VTP) required for targeting to the vacuole, which is removed by proteolytic cleavage. We investigated transformed N. sylvestris plants constitutively expressing CHN A or a mutant CHN A lacking the chitin-binding domain and spacer (ΔCS CHN A), as well as N. plumbaginifolia protoplasts transiently expressing the same constructs. Processing and transport in the two systems was very similar. A shift in the apparent molecular weight of chitinase, indicative of prolyl hydroxylation, was detectable only 30 min after appearance of newly synthesized prochitinase, indicating that it might occur in a post-ER compartment. In total, labelled chitinase was detected in the microsomal fraction for up to 90–120 min as a prochitinase, bearing the VTP. Later, it appeared only in the soluble fraction (comprising the vacuolar sap) as the mature CHN A without the VTP. In both systems, intracellular transport and processing of ΔCS CHN A was faster than that of the wildtype form, indicating that correct folding of the cysteine-rich chitin-binding domain and/or prolyl hydroxylation of the spacer delays transport to the vacuole.