Instantaneous Flow Analysis of Contractile Cytoskeletal Structures Affected by the Dysregulation of Kinesin and Tropomyosin Motors

ABSTRACT The complex dynamics of cytoskeletal meshworks make them a difficult subject of study. With the advent of fluorescent speckle microscopy (FSM) and other technological advances in microscopy techniques, much more is now known about how the filamentous actin (F-actin) and MT networks work within cells to give rise to the vast array of functions which require them. A current challenge to the imaging field is to improve the utility and accuracy of the computational approaches required to analyze large and complex imaging datasets. Here, we present the results of a computational method that, when applied to FSM time-lapse series, can capture the instantaneous state of the rapidly changing, dense, and multi-directional speckle flows often exhibited by cytoskeletal dynamics in living systems. Re-analysis of previously published FSM image sets demonstrates that this method, which we call the Instantaneous Flow Tracking Algorithm (IFTA), can accurately detect speckle flows in mitotic spindles and F-actin meshworks, even in regions of high dynamicity of overlapping, anti-parallel flows where previous methods failed. The anti-parallel flow at the metaphase plate of the mitotic spindle is a well-known phenomenon during the initial stages of chromosome segregation and it has been measured by several approaches, mostly in stationary spindles which do not exhibit any polar rotation. The mitotic spindle is the target of many cancer and neurodegenerative drugs and as such, there has been many attempts at inhibiting its basic functions with the objective of preventing chromosome segregation and the formation of new daughter cells. Such attempts have largely been focused on the inhibition of the action of MT plus-end directed motors, for instance the kinesin Eg5. Spindles with inhibited kinesins have been thought to exhibit no MT flux, however IFTA measured regional flux of up to 2.7 µm/min, which reveals the activity of potent secondary flux mechanisms. These results suggest novel, additional, approaches toward arresting cells in mitosis during patient treatment. The traditional tracking methods erroneously measure zero flux in areas where contractile F-actin flows meet, denoted as a “convergence zone” and commonly found in the lamella of motile cells and the neck of growth cones. When IFTA was used to analyze FSM datasets derived from these structures, we detected high rates of protein turnover, anti-parallel speckle motion, and fast flux of actin subunits in both directions in the same “convergence zones”. This demonstrates the presence of a molecular machinery based on contractility in the lamella/lamellipodium of migrating cells and at the base of growing neurons, which can be exploited in the clinic. When applied to FSM data of migrating kangaroo rat kidney epithelial Ptk1 cells over-expressing different isoforms of the actin-based motor tropomyosin, IFTA revealed distinct, isoform-specific effects on contractile F-actin flows. Specifically, we found that decreased affinity between tropomyosin and F-actin correlated with an increase in speckle velocity heterogeneity. Such quantitative imaging analysis offers the ability to reliably test novel therapeutics ex vivo . In summary, our results demonstrate that IFTA is a valuable tool that, in contrast to other existing trackers, can accurately resolve the complex, yet organized dynamics of interconnected polymers of cytoskeleton proteins, such as tubulin and actin.