Mammalian Navigators are microtubule plus-end tracking proteins that can reorganize the cytoskeleton to induce neurite-like extensions.

Mammalian microtubule plus-end tracking proteins (+TIPs) specifically associate with the ends of growing microtubules. +TIPs are involved in many cellular processes, including mitosis, cell migration and neurite extension. Navigators are mammalian homologues of the C. elegans unc-53 protein, an ATPase that has been linked to the migration and outgrowth of muscles, axons and excretory canals. Here we show that all three mammalian Navigators are +TIPs, consistent with a previous study on Navigator 1 (NAV1) (Martinez-Lopez et al., Mol Cell Neurosci 2005;28:599-612). Overexpression of GFP-tagged Navigators causes displacement of CAP_GLY-motif containing +TIPs, such as CLIP-170, from microtubule ends, suggesting that the Navigator-binding sites on microtubule ends overlap with those of the CAP_GLY-motif proteins. In interphase cells, mammalian Navigators also prominently localize to centrosomes, a localization that does not depend on an intact microtubule network. Fluorescence recovery after photobleaching (FRAP) experiments indicate that NAV1 associates with intracellular structures other than microtubules or centrosomes. Expression of GFP-tagged Navigators induces the formation of neurite-like extensions in non-neuronal cells, showing that Navigators can dominantly alter cytoskeletal behavior. For NAV1 this function depends on its ATPase activity; it is not achieved by a classical type of MT bundling and stabilization. Combined our data suggest that Navigators are +TIPs that can reorganize the cytoskeleton to guide cell shape changes. Our data are consistent with a role for Navigators in neurite outgrowth.

Particle filtering for multiple object tracking in dynamic fluorescence microscopy images: application to microtubule growth analysis.

Quantitative analysis of dynamic processes in living cells by means of fluorescence microscopy imaging requires tracking of hundreds of bright spots in noisy image sequences. Deterministic approaches, which use object detection prior to tracking, perform poorly in the case of noisy image data. We propose an improved, completely automatic tracker, built within a Bayesian probabilistic framework. It better exploits spatiotemporal information and prior knowledge than common approaches, yielding more robust tracking also in cases of photobleaching and object interaction. The tracking method was evaluated using simulated but realistic image sequences, for which ground truth was available. The results of these experiments show that the method is more accurate and robust than popular tracking methods. In addition, validation experiments were conducted with real fluorescence microscopy image data acquired for microtubule growth analysis. These demonstrate that the method yields results that are in good agreement with manual tracking performed by expert cell biologists. Our findings suggest that the method may replace laborious manual procedures.

Rao-Blackwellized marginal particle filtering for multiple object tracking in molecular bioimaging.

Modern live cell fluorescence microscopy imaging systems, used abundantly for studying intra-cellular processes in vivo, generate vast amounts of noisy image data that cannot be processed efficiently and accurately by means of manual or current computerized techniques. We propose an improved tracking method, built within a Bayesian probabilistic framework, which better exploits temporal information and prior knowledge. Experiments on simulated and real fluorescence microscopy image data acquired for microtubule dynamics studies show that the technique is more robust to noise, photobleaching, and object interaction than common tracking methods and yields results that are in good agreement with expert cell biologists.

Role of CLASP2 in microtubule stabilization and the regulation of persistent motility.

In motile fibroblasts, stable microtubules (MTs) are oriented toward the leading edge of cells. How these polarized MT arrays are established and maintained, and the cellular processes they control, have been the subject of many investigations. Several MT "plus-end-tracking proteins," or +TIPs, have been proposed to regulate selective MT stabilization, including the CLASPs, a complex of CLIP-170, IQGAP1, activated Cdc42 or Rac1, a complex of APC, EB1, and mDia1, and the actin-MT crosslinking factor ACF7. By using mouse embryonic fibroblasts (MEFs) in a wound-healing assay, we show here that CLASP2 is required for the formation of a stable, polarized MT array but that CLIP-170 and an APC-EB1 interaction are not essential. Persistent motility is also hampered in CLASP2-deficient MEFs. We find that ACF7 regulates cortical CLASP localization in HeLa cells, indicating it acts upstream of CLASP2. Fluorescence-based approaches show that GFP-CLASP2 is immobilized in a bimodal manner in regions near cell edges. Our results suggest that the regional immobilization of CLASP2 allows MT stabilization and promotes directionally persistent motility in fibroblasts.