Microtubule dynamics influence the retrograde biased motility of kinesin-4 motor teams in neuronal dendrites.

Microtubules establish the directionality of intracellular transport by kinesins and dynein through polarized assembly, but it remains unclear how directed transport occurs along microtubules organized with mixed polarity. We investigated the ability of the plus end-directed kinesin-4 motor KIF21B to navigate mixed polarity microtubules in mammalian dendrites. Reconstitution assays with recombinant KIF21B and engineered microtubule bundles or extracted neuronal cytoskeletons indicate that nucleotide-independent microtubule-binding regions of KIF21B modulate microtubule dynamics and promote directional switching on antiparallel microtubules. Optogenetic recruitment of KIF21B to organelles in live neurons induces unidirectional transport in axons but bidirectional transport with a net retrograde bias in dendrites. Removal of the secondary microtubule-binding regions of KIF21B or dampening of microtubule dynamics with low concentrations of nocodazole eliminates retrograde bias in live dendrites. Further exploration of the contribution of microtubule dynamics in dendrites to directionality revealed plus end-out microtubules to be more dynamic than plus end-in microtubules, with nocodazole preferentially stabilizing the plus end-out population. We propose a model in which both nucleotide-sensitive and -insensitive microtubule-binding sites of KIF21B motors contribute to the search and selection of stable plus end-in microtubules within the mixed polarity microtubule arrays characteristic of mammalian dendrites to achieve net retrograde movement of KIF21B-bound cargoes.

Cega: a single particle segmentation algorithm to identify moving particles in a noisy system.

Improvements to particle tracking algorithms are required to effectively analyze the motility of biological molecules in complex or noisy systems. A typical single particle tracking (SPT) algorithm detects particle coordinates for trajectory assembly. However, particle detection filters fail for data sets with low signal-to-noise levels. When tracking molecular motors in complex systems, standard techniques often fail to separate the fluorescent signatures of moving particles from background signal. We developed an approach to analyze the motility of kinesin motor proteins moving along the microtubule cytoskeleton of extracted neurons using the Kullback-Leibler divergence to identify regions where there are significant differences between models of moving particles and background signal. We tested our software on both simulated and experimental data and found a noticeable improvement in SPT capability and a higher identification rate of motors as compared with current methods. This algorithm, called Cega, for "find the object," produces data amenable to conventional blob detection techniques that can then be used to obtain coordinates for downstream SPT processing. We anticipate that this algorithm will be useful for those interested in tracking moving particles in complex in vitro or in vivo environments.

Differential mast cell outcomes are sensitive to FcεRI-Syk binding kinetics.

Cross-linking of immunoglobulin E-bound FcεRI triggers multiple cellular responses, including degranulation and cytokine production. Signaling is dependent on recruitment of Syk via docking of its dual SH2 domains to phosphorylated tyrosines within the FcεRI immunoreceptor tyrosine-based activation motifs. Using single-molecule imaging in live cells, we directly visualized and quantified the binding of individual mNeonGreen-tagged Syk molecules as they associated with the plasma membrane after FcεRI activation. We found that Syk colocalizes transiently to FcεRI and that Syk-FcεRI binding dynamics are independent of receptor aggregate size. Substitution of glutamic acid for tyrosine between the Syk SH2 domains (Syk-Y130E) led to an increased Syk-FcεRI off-rate, loss of site-specific Syk autophosphorylation, and impaired downstream signaling. Genome edited cells expressing only Syk-Y130E were deficient in antigen-stimulated calcium release, degranulation, and production of some cytokines (TNF-a, IL-3) but not others (MCP-1, IL-4). We propose that kinetic discrimination along the FcεRI signaling pathway occurs at the level of Syk-FcεRI interactions, with key outcomes dependent upon sufficiently long-lived Syk binding events.

Estimation of the diffusion constant from intermittent trajectories with variable position uncertainties.

The movement of a particle described by Brownian motion is quantified by a single parameter, D, the diffusion constant. The estimation of D from a discrete sequence of noisy observations is a fundamental problem in biological single-particle tracking experiments since it can provide information on the environment and/or the state of the particle itself via the hydrodynamic radius. Here, we present a method to estimate D that takes into account several effects that occur in practice, important for the correct estimation of D, and that have hitherto not been combined together for an estimation of D. These effects are motion blur from the finite integration time of the camera, intermittent trajectories, and time-dependent localization uncertainty. Our estimation procedure, a maximum-likelihood estimation with an information-based confidence interval, follows directly from the likelihood expression for a discretely observed Brownian trajectory that explicitly includes these effects. We begin with the formulation of the likelihood expression and then present three methods to find the exact solution. Each method has its own advantages in either computational robustness, theoretical insight, or the estimation of hidden variables. The Fisher information for this likelihood distribution is calculated and analyzed to show that localization uncertainties impose a lower bound on the estimation of D. Confidence intervals are established and then used to evaluate our estimator on simulated data with experimentally relevant camera effects to demonstrate the benefit of incorporating variable localization errors.

Publisher's Note: Estimation of the diffusion constant from intermittent trajectories with variable position uncertainties [Phys. Rev. E 93, 042401 (2016)].

This corrects the article DOI: 10.1103/PhysRevE.93.042401.