Interplay between Toxin Transport and Flotillin Localization

The flotillin proteins are localized in lipid domains at the plasma membrane as well as in intracellular compartments. In the present study, we examined the importance of flotillin-1 and flotillin-2 for the uptake and transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin and we investigated whether toxin binding and uptake were associated with flotillin relocalization. We observed a toxin-induced redistribution of the flotillins, which seemed to be regulated in a p38-dependent manner. Our experiments provide no evidence for a changed endocytic uptake of Stx or ricin in cells silenced for flotillin-1 or -2. However, the Golgi-dependent sulfation of both toxins was significantly reduced in flotillin knockdown cells. Interestingly, when the transport of ricin to the ER was investigated, we obtained an increased mannosylation of ricin in flotillin-1 and flotillin-2 knockdown cells. The toxicity of both toxins was twofold increased in flotillin-depleted cells. Since BFA (Brefeldin A) inhibits the toxicity even in flotillin knockdown cells, the retrograde toxin transport is apparently still Golgi-dependent. Thus, flotillin proteins regulate and facilitate the retrograde transport of Stx and ricin.     

A 3D Hybrid Model for Tissue Growth: The Interplay between Cell Population and Mass Transport Dynamics

To provide theoretical guidance for the design and in vitro cultivation of bioartificial tissues, we have developed a multiscale computational model that can describe the complex interplay between cell population and mass transport dynamics that governs the growth of tissues in three-dimensional scaffolds. The model has three components: a transient partial differential equation for the simultaneous diffusion and consumption of a limiting nutrient; a cellular automaton describing cell migration, proliferation, and collision; and equations that quantify how the varying nutrient concentration modulates cell division and migration. The hybrid discrete-continuous model was parallelized and solved on a distributed-memory multicomputer to study how transport limitations affect tissue regeneration rates under conditions encountered in typical bioreactors. Simulation results show that the severity of transport limitations can be estimated by the magnitude of two dimensionless groups: the Thiele modulus and the Biot number. Key parameters including the initial seeding mode, cell migration speed, and the hydrodynamic conditions in the bioreactor are shown to affect not only the overall rate, but also the pattern of tissue growth. This study lays the groundwork for more comprehensive models that can handle mixed cell cultures, multiple nutrients and growth factors, and other cellular processes, such as cell death.     

Untangling the Interplay between Epidemic Spread and Transmission Network Dynamics

The epidemic spread of infectious diseases is ubiquitous and often has a considerable impact on public health and economic wealth. The large variability in the spatio-temporal patterns of epidemics prohibits simple interventions and requires a detailed analysis of each epidemic with respect to its infectious agent and the corresponding routes of transmission. To facilitate this analysis, we introduce a mathematical framework which links epidemic patterns to the topology and dynamics of the underlying transmission network. The evolution, both in disease prevalence and transmission network topology, is derived from a closed set of partial differential equations for infections without allowing for recovery. The predictions are in excellent agreement with complementarily conducted agent-based simulations. The capacity of this new method is demonstrated in several case studies on HIV epidemics in synthetic populations: it allows us to monitor the evolution of contact behavior among healthy and infected individuals and the contributions of different disease stages to the spreading of the epidemic. This gives both direction to and a test bed for targeted intervention strategies for epidemic control. In conclusion, this mathematical framework provides a capable toolbox for the analysis of epidemics from first principles. This allows for fast, in silico modeling--and manipulation--of epidemics and is especially powerful if complemented with adequate empirical data for parameterization.     

Interplay between Velocity and Travel Distance of Kinesin-based Transport in the Presence of Tau

Although the disease-relevant microtubule-associated protein tau is known to severely inhibit kinesin-based transport in vitro, the potential mechanisms for reversing this detrimental effect to maintain healthy transport in cells remain unknown. Here we report the unambiguous upregulation of multiple-kinesin travel distance despite the presence of tau, via decreased single-kinesin velocity. Interestingly, the presence of tau also modestly reduced cargo velocity in multiple-kinesin transport, and our stochastic simulations indicate that the tau-mediated reduction in single-kinesin travel underlies this observation. Taken together, our observations highlight a nontrivial interplay between velocity and travel distance for kinesin transport, and suggest that single-kinesin velocity is a promising experimental handle for tuning the effect of tau on multiple-kinesin travel distance.     

Interplay between Velocity and Travel Distance of Kinesin-based Transport in the Presence of Tau

Although the disease-relevant microtubule-associated protein tau is known to severely inhibit kinesin-based transport in vitro, the potential mechanisms for reversing this detrimental effect to maintain healthy transport in cells remain unknown. Here we report the unambiguous upregulation of multiple-kinesin travel distance despite the presence of tau, via decreased single-kinesin velocity. Interestingly, the presence of tau also modestly reduced cargo velocity in multiple-kinesin transport, and our stochastic simulations indicate that the tau-mediated reduction in single-kinesin travel underlies this observation. Taken together, our observations highlight a nontrivial interplay between velocity and travel distance for kinesin transport, and suggest that single-kinesin velocity is a promising experimental handle for tuning the effect of tau on multiple-kinesin travel distance.Conventional kinesin is a major microtubule-based molecular motor that enables long-range transport in living cells. Although traditionally investigated in the context of single-motor experiments, two or more kinesin motors are often linked together to transport the same cargo in vivo (1–4). Understanding the control and regulation of the group function of multiple kinesins has important implications for reversing failure modes of transport in a variety of human diseases, particularly neurodegenerative diseases. Tau is a disease-relevant protein enriched in neurons (5,6). The decoration of microtubules with tau is known to strongly inhibit kinesin transport in vitro (7–9), but how kinesin-based transport is maintained in the presence of high levels of tau, particularly in healthy neurons, remains an important open question. To date, no mechanism has been directly demonstrated to reverse the inhibitory effect of tau on kinesin-based transport. Here we present a simple in vitro study that demonstrates the significant upregulation of multiple-kinesin travel distance with decreasing ATP concentration, despite the presence of tau.This investigation was motivated by our recent finding that single-kinesin velocity is a key controller for multiple-kinesin travel distance along bare microtubules (10). The active stepping of each kinesin motor is stimulated by ATP (11), and each kinesin motor remains strongly bound to the microtubule between successive steps (10,11). As demonstrated for bare microtubules (10), with decreasing ATP concentrations, each microtubule-bound kinesin experiences a decreased stepping rate per unit time and spends an increased fraction of time in the strongly bound state; additional unbound kinesins on the same cargo have more time to bind to the microtubule before cargo travel terminates. Thus, reductions in single-kinesin velocity increase the probability that at least one kinesin motor will remain bound to the microtubule per unit time, thereby increasing the travel distance of each cargo (10). Because this effect only pertains to the stepping rate of each individual kinesin and does not address the potential presence of roadblocks such as tau on the microtubules, we hypothesized in this study that single-kinesin velocity may be exploited to relieve the impact of tau on multiple-kinesin travel distance.We focused our in vitro investigation on human tau 23 (htau23, or 3RS tau), an isoform of tau that exhibits the strongest inhibitory effect on kinesin-based transport (7–9). Importantly, htau23 does not alter the stepping rate of individual kinesins (7,9), supporting our hypothesis and enabling us to decouple single-kinesin velocity from the potential effects of tau. We carried out multiple-kinesin motility experiments using polystyrene beads as in vitro cargos (8,10), ATP concentration as an in vitro handle to controllably tune single-kinesin velocity (10,11), and three input kinesin concentrations to test the generality of potential findings for multiple-kinesin transport. Combined with previous two-kinesin studies (10,12), our measurements of travel distance (Fig. 1 A) indicate that the lowest kinesin concentration employed (0.8 nM) corresponds to an average of ∼2–3 kinesins per cargo. Note that in the absence of tau, the observed decrease in bead velocity at the higher kinesin concentrations (Fig. 1 A) is consistent with a recent in vitro finding (13). At 1 mM ATP, htau23 reduced kinesin-based travel distance by a factor of two or more (Fig. 1, A and B). This observation is in good agreement with previous reports (7,8).Open in a separate windowFigure 1Distributions of multiple-kinesin travel distances measured at three experimental conditions, to verify the effect of tau (A and B) and to investigate the impact of single-kinesin velocity on the tau effect (B and C). Shaded bars at 8.7 μm indicate counts of travel exceeding the field of view. The mean travel distance (d; ± standard error of mean, SEM), sample size (n), and corresponding mean velocity (v; ± SEM) are indicated. MT denotes microtubule. Mean travel distance increased substantially at 20 μM ATP (C), despite the presence of htau23. This effect persisted across all three kinesin concentrations tested (left to right).Consistent with our hypothesis, reducing the available ATP concentration to 20 μM increased the multiple-kinesin travel distance by >1.4-fold for all three input kinesin concentrations (Fig. 1, B and C), despite the presence of htau23. The corresponding reduction in single-kinesin velocity with decreasing ATP concentration (10,11) is reflected in the ∼3.4-fold reduction in the measured bead velocities (Fig. 1, B and C). Therefore, the strong negative relationship between single-kinesin velocity and multiple-kinesin travel distance occurs not only for bare microtubules (10), but also for tau-decorated microtubules.What causes the observed increase in travel distance at the lower ATP concentration (Fig. 1, B and C)? In addition to the mechanism discussed above for the case of bare microtubules (10), an intriguing mechanism was suggested by recent studies of tau-microtubule interactions in which htau23 was observed to dynamically diffuse along microtubule lattices (14,15): reducing the stepping rate of a microtubule-bound kinesin may effectively increase the probability that a tau roadblock can diffuse away before the kinesin takes its next step.Perhaps surprisingly, although htau23 does not impact single-kinesin velocity (7,9), we observed a modest reduction in the average velocity of multiple-kinesin transport in experiments using tau-decorated microtubules (Fig. 1, A and B). This decreased velocity reflects a substantially larger variance in the instantaneous velocity for bead trajectories in the presence of htau23 (see Fig. S1 in the Supporting Material), as quantified by parsing each bead trajectory into a series of constant-velocity segments using a previously developed automatic software incorporating Bayesian statistics (16).To test the possibility that single-kinesin travel distance impacts multiple-kinesin velocity, we performed stochastic simulations (see the Supporting Material) that assumed N identical kinesin motors available for transport and included kinesin’s detachment kinetics (17). Previously, this model successfully captured multiple-dynein travel distances in vivo using single-dynein characteristics measured in vitro (18). In this study, we introduced one (and only one) free parameter to reflect the probability of each bound kinesin encountering tau at each step. When encountering tau, each kinesin has a 54% probability of detaching from the microtubule (interpolated from Fig. 2A of Dixit et al. (7)); the undetached kinesin is assumed to remain engaged in transport and completes its step along the microtubule despite the presence of tau.Remarkably, our simple simulation suggested that the tau-mediated reduction in single-kinesin travel is sufficient to reduce multiple-kinesin velocity (Fig. 2 A). The majority of the velocity decrease is predicted to occur at the transition from single-kinesin to two-kinesin transport (Fig. 2). Further decreases in cargo velocity with increasing motor number are predicted to be modest and largely independent of tau (Fig. 2 B). The results of our simulation remain qualitatively the same when evaluated at two bounds (40 and 65%) encompassing the interpolated 54% probability of kinesin detaching at tau (see Fig. S2).Open in a separate windowFigure 2Stochastic simulations predict a tau-dependent reduction in multiple-kinesin velocity, assuming that the only effect of tau protein is to prematurely detach kinesin from the microtubule (or, to reduce single-kinesin travel distance). (A) Average velocity of cargos carried by the indicated number of kinesins was evaluated at 1 mM ATP, and for four probabilities that a kinesin may encounter tau at each step. Mean velocity was evaluated using 600 simulated trajectories for all simulation conditions. Error bars indicate SEM. (B) Change in cargo velocity with each additional kinesin (ΔVel/kinesin) as a function of tau-encounter probability. These values were calculated from cargo velocities shown in panel A. Error bars indicate SEM.We note that our simple simulations do not consider the possibility that kinesin may pause in front of a tau roadblock, as previously reported in Dixit et al. (7). We omitted this consideration because the interaction strength between kinesin and the microtubule in such a paused state is unknown. In a multiple-motor geometry, could a paused kinesin be dragged along by the other motors bound to the same cargo? Could a tau roadblock be forcefully swept off the microtubule surface by the collective motion of the cargo-motor complex? Significant experimental innovations are necessary to specifically address these questions in future multiple-motor assays and to guide modeling efforts. Nonetheless, our simple simulation demonstrates that reducing single-kinesin travel distance is sufficient to decrease multiple-kinesin travel distance.Taken together, our observations highlight a nontrivial interplay between velocity and travel distance for kinesin-based transport in the presence of tau. We uncover a previously unexplored dual inhibition of tau on kinesin-transport: in addition to limiting cargo travel distance, the tau-mediated reduction in single-kinesin travel distance also leads to a modest reduction in multiple-kinesin velocity. We provide what we believe to be the first demonstration of the unambiguous upregulation of multiple-kinesin travel distance despite the presence of tau, via reducing single-kinesin velocity, suggesting a mechanism that could be harnessed for future therapeutic interventions in diseases that result from aberrant kinesin-based transport.     

Interplay between Bimolecular Recombination and Carrier Transport Distances in Bulk Heterojunction Organic Solar Cells

In this work, it is demonstrated that bimolecular recombination depends on the distance that free carriers are required to travel in transit to the electrodes in bulk heterojunction organic solar cells. By employing semi‐transparent devices, the carrier transport distance can be controlled via the local light absorption profile with an appropriate choice of the illumination side and incident wavelength. Using a series of light intensity‐dependent measurements, bimolecular recombination is shown to depend on the distance electrons or holes are required to transit the active layer. This effect is demonstrated for three different bulk heterojunction blends, where the restrictive carrier that causes the onset of recombination is identified. The mobility‐lifetime products of the limiting carriers are also estimated using a simple model for carrier extraction, where similar values are obtained regardless of the absorption profile. Implications for 1‐sun performance are also discussed, which provide guidelines for fabricating devices with thicker active layers capable of maximizing light absorption without succumbing to recombination losses.     

Effects of Plectin Depletion on Keratin Network Dynamics and Organization

The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin β4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and β1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells.     

Kindlin-1 Regulates Integrin Dynamics and Adhesion Turnover

Loss-of-function mutations in the gene encoding the integrin co-activator kindlin-1 cause Kindler syndrome. We report a novel kindlin-1-deficient keratinocyte cell line derived from a Kindler syndrome patient. Despite the expression of kindlin-2, the patient’s cells display several hallmarks related to reduced function of β1 integrins, including abnormal cell morphology, cell adhesion, cell spreading, focal adhesion assembly, and cell migration. Defective cell adhesion was aggravated by kindlin-2 depletion, indicating that kindlin-2 can compensate to a certain extent for the loss of kindlin-1. Intriguingly, β1 at the cell-surface was aberrantly glycosylated in the patient’s cells, and its expression was considerably reduced, both in cells in vitro and in the patient’s epidermis. Reconstitution with wild-type kindlin-1 but not with a β1-binding defective mutant restored the aberrant β1 expression and glycosylation, and normalized cell morphology, adhesion, spreading, and migration. Furthermore, the expression of wild-type kindlin-1, but not of the integrin-binding-defective mutant, increased the stability of integrin-mediated cell-matrix adhesions and enhanced the redistribution of internalized integrins to the cell surface. Thus, these data uncover a role for kindlin-1 in the regulation of integrin trafficking and adhesion turnover.     

Interplay between inflammation and cancer

Tumor-promoting inflammation is one of the hallmarks of cancer. It has been shown that cancer development is strongly influenced by both chronic and acute inflammation process. Progress in research on inflammation revealed a connection between inflammatory processes and neoplastic transformation, the progression of tumour, and the development of metastases and recurrences. Moreover, the tumour invasive procedures (both surgery and biopsy) affect the remaining tumour cells by increasing their survival, proliferation and migration. One of the concepts explaining this phenomena is an induction of a wound healing response. While in normal tissue it is necessary for tissue repair, in tumour tissue, induction of adaptive and innate immune response related to wound healing, stimulates tumour cell survival, angiogenesis and extravasation of circulating tumour cells. It has become evident that certain types of immune response and immune cells can promote tumour progression more than others. In this review, we focus on current knowledge on carcinogenesis and promotion of cancer growth induced by inflammatory processes.     

Interplay between wrn and the checkpoint in s-phase

Stability of the genome is crucial for survival and faithful transmission of the genetic blueprint to progenitors. During DNA replication chromosome integrity can be challenged by a variety of exogenous and endogenous damaging agents and by the process of duplication itself. Thus, eukaryotic cells have evolved a sophisticated response called replication checkpoint supervising the accurate and complete genome replication. The replication checkpoint response bridges together replication, repair and cell cycle proteins in a coordinated network having the ATR kinase as culprit. ATR-mediated phosphorylation events control that stalled replication forks are properly sensed and stabilised, cell cycle progression halted and replication eventually recovered. In the recent years, the Werner syndrome protein (WRN) emerged as a central actor of the replication checkpoint being instrumental for correct recovery from arrested replication and a substrate of ATR. In this review, how WRN and the replication checkpoint could cross-talk and contribute to faithful recovery of stalled replication forks will be discussed.     

Interplay between membrane curvature and the actin cytoskeleton

An intimate interplay of the plasma membrane with curvature-sensing and curvature-inducing proteins would allow for defining specific sites or nanodomains of action at the plasma membrane, for example, for protrusion, invagination, and polarization. In addition, such connections are predestined to ensure spatial and temporal order and sequences. The combined forces of membrane shapers and the cortical actin cytoskeleton might hereby in particular be required to overcome the strong resistance against membrane rearrangements in case of high plasma membrane tension or cellular turgor. Interestingly, also the opposite might be necessary, the inhibition of both membrane shapers and cytoskeletal reinforcement structures to relieve membrane tension to protect cells from membrane damage and rupturing during mechanical stress. In this review article, we discuss recent conceptual advances enlightening the interplay of plasma membrane curvature and the cortical actin cytoskeleton during endocytosis, modulations of membrane tensions, and the shaping of entire cells.     

Quantitative Analysis of Ezrin Turnover Dynamics in the Actin Cortex

Proteins of the ERM family (ezrin, moesin, radixin) play a fundamental role in tethering the membrane to the cellular actin cortex as well as regulating cortical organization and mechanics. Overexpression of dominant inactive forms of ezrin leads to fragilization of the membrane-cortex link and depletion of moesin results in softer cortices that disrupt spindle orientation during cytokinesis. Therefore, the kinetics of association of ERM proteins with the cortex likely influence the timescale of cortical signaling events and the dynamics of membrane interfacing to the cortex. However, little is known about ERM protein turnover at the membrane-cortex interface. Here, we examined cortical ezrin dynamics using fluorescence recovery after photobleaching experiments and single-molecule imaging. Using multiexponential fitting of fluorescence recovery curves, we showed that ezrin turnover resulted from three molecular mechanisms acting on very different timescales. The fastest turnover process was due to association/dissociation from the F-actin cortex, suggesting that ezrin acts as a link that leads to low friction between the membrane and the cortex. The second turnover process resulted from association/dissociation of ezrin from the membrane and the slowest turnover process resulted from the slow diffusion of ezrin in the plane of the membrane. In summary, ezrin-mediated membrane-cortex tethering resulted from long-lived interactions with the membrane via the FERM domain coupled with shorter-lived interactions with the cortex. The slow diffusion of membranous ezrin and its interaction partners relative to the cortex signified that signals emanating from membrane-associated ezrin may locally act to modulate cortical organization and contractility.     

Quantitative Analysis of Ezrin Turnover Dynamics in the Actin Cortex

Proteins of the ERM family (ezrin, moesin, radixin) play a fundamental role in tethering the membrane to the cellular actin cortex as well as regulating cortical organization and mechanics. Overexpression of dominant inactive forms of ezrin leads to fragilization of the membrane-cortex link and depletion of moesin results in softer cortices that disrupt spindle orientation during cytokinesis. Therefore, the kinetics of association of ERM proteins with the cortex likely influence the timescale of cortical signaling events and the dynamics of membrane interfacing to the cortex. However, little is known about ERM protein turnover at the membrane-cortex interface. Here, we examined cortical ezrin dynamics using fluorescence recovery after photobleaching experiments and single-molecule imaging. Using multiexponential fitting of fluorescence recovery curves, we showed that ezrin turnover resulted from three molecular mechanisms acting on very different timescales. The fastest turnover process was due to association/dissociation from the F-actin cortex, suggesting that ezrin acts as a link that leads to low friction between the membrane and the cortex. The second turnover process resulted from association/dissociation of ezrin from the membrane and the slowest turnover process resulted from the slow diffusion of ezrin in the plane of the membrane. In summary, ezrin-mediated membrane-cortex tethering resulted from long-lived interactions with the membrane via the FERM domain coupled with shorter-lived interactions with the cortex. The slow diffusion of membranous ezrin and its interaction partners relative to the cortex signified that signals emanating from membrane-associated ezrin may locally act to modulate cortical organization and contractility.