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We measured actin turnover in lamellipodia and lamellae of migrating cells, using quantitative Fluorescent Speckle Microscopy. Lamellae disassembled at low rates from the front to the back. However, the dominant feature in their turnover was a spatially random pattern of periodic polymerization and depolymerization moving with the retrograde flow. Power spectra contained frequencies between 0.5 and 1 cycle/min. The spectra remained unchanged when applying Latrunculin A and Jasplakinolide in low doses, except that additional frequencies occurred beyond 1 cycle/min. Whereas Latrunculin did not change the rate of mean disassembly, Jasplakinolide halted it completely, indicating that the steady state and the dynamics of actin turnover are differentially affected by pharmacological agents. Lamellipodia assembled in recurring bursts at the leading edge and disassembled approximately 2.5 microm behind. Events of polymerization correlated spatially and temporally with transient formation of Arp2/3 clusters. In lamellae, Arp2/3 accumulation and polymerization correlated only spatially, suggesting an Arp2/3-independent mechanism for filament nucleation. To acquire these data we had to enhance the resolution of quantitative Fluorescent Speckle Microscopy to the submicron level. Several algorithmic advances in speckle image processing are described enabling the analysis of kinetic and kinematic activities of polymer networks at the level of single speckles.

Fluorescent speckle microscopy (FSM) is becoming the technique of choice for analyzing in vivo the dynamics of polymer assemblies, such as the cytoskeleton. The massive amount of data produced by this method calls for computational approaches to recover the quantities of interest; namely, the polymerization and depolymerization activities and the motions undergone by the cytoskeleton over time. Attempts toward this goal have been hampered by the limited signal-to-noise ratio of typical FSM data, by the constant appearance and disappearance of speckles due to polymer turnover, and by the presence of flow singularities characteristic of many cytoskeletal polymer assemblies. To deal with these problems, we present a particle-based method for tracking fluorescent speckles in time-lapse FSM image series, based on ideas from operational research and graph theory. Our software delivers the displacements of thousands of speckles between consecutive frames, taking into account that speckles may appear and disappear. In this article we exploit this information to recover the speckle flow field. First, the software is tested on synthetic data to validate our methods. We then apply it to mapping filamentous actin retrograde flow at the front edge of migrating newt lung epithelial cells. Our results confirm findings from previously published kymograph analyses and manual tracking of such FSM data and illustrate the power of automated tracking for generating complete and quantitative flow measurements. Third, we analyze microtubule poleward flux in mitotic metaphase spindles assembled in Xenopus egg extracts, bringing new insight into the dynamics of microtubule assemblies in this system.

Fluorescent speckle microscopy (FSM) is a new imaging technique with the potential for simultaneous visualization of translocation and dynamic turnover of polymer structures. However, the use of FSM has been limited by the lack of specialized software for analysis of the positional and photometric fluctuations of hundreds of thousand speckles in an FSM time-lapse series, and for translating this data into biologically relevant information. In this paper we present a first version of a software for automated analysis of FSM movies. We focus on mapping the assembly and disassembly kinetics of a polymer meshwork. As a model system we have employed cortical F-actin meshworks in live newt lung epithelial cells. We lay out the algorithm in detail and present results of our analysis. The high spatial and temporal resolution of our maps reveals a kinetic cycling of F-actin, where phases of polymerization alternate with depolymerization in a spatially coordinated fashion. The cycle rates change when treating cells with a low dose of the drug latrunculin A. This shows the potential of this technique for future quantitative screening of drugs affecting the actin cytoskeleton. Various control experiments demonstrate that the algorithm is robust with respect to intensity variations due to noise and photobleaching and that effects of focus plane drifts can be eliminated by manual refocusing during image acquisition.

Migrating cells display a characteristic polarization of the actin cytoskeleton. Actin filaments polymerise in the protruding front of the cell whereas actin filament bundles contract in the cell body, which results in retraction of the cell's rear. The dynamic organization of the actin cytoskeleton provides the force for cell motility and is regulated by small GTPases of the Rho family, in particular Rac1, RhoA and Cdc42. Although the microtubule cytoskeleton is also polarized in a migrating cell, and microtubules are essential for the directed migration of many cell types, their role in cell motility is not well understood at a molecular level. Here, we discuss the potential molecular mechanisms for interplay of microtubules, actin and Rho GTPase signalling in cell polarization and motility. Recent evidence suggests that microtubules locally modulate the activity of Rho GTPases and, conversely, Rho GTPases might be responsible for the initial polarization of the microtubule cytoskeleton. Thus, microtubules might be part of a positive feedback mechanism that maintains the stable polarization of a directionally migrating cell.

Microtubule-associated proteins (MAPs) are proteins that reversibly bind to and regulate microtubule dynamics and functions in vivo. We examined the dynamics of binding of a MAP called ensconsin (E-MAP-115) to microtubules in vivo. We used 5xGFP-EMTB, a construct in which the microtubule-binding domain of ensconsin (EMTB) is fused to five copies of green fluorescent protein (GFP), as a reporter molecule amenable to the use of fluorescent speckle microscopy. Fluorescent speckle microscopy (FSM) sequences and kymograph analyses showed rapid dynamics of speckles comprised of 5xGFP-EMTB in untreated cells. By contrast, in detergent-lysed cytoskeletons, speckles were not dynamic. Since detergent-lysed cytoskeletons differ from living cells in that they lack both ATP and dynamic microtubules, we used azide treatment to substantially reduce the level of ATP in living cells and we used Taxol to halt microtubule dynamics. Both treatments slowed the dynamics of 5xGFP-EMTB speckles observed by FSM. We also used fluorescence recovery after photobleaching (FRAP) to quantify the half-time of binding and dissociation of the 5xGFP-EMTB chimera and to compare this half-time to that of the full-length MAP molecule. In untreated cells, the t(g) of either 5xGFP-EMTB or full-length GFP-ensconsin was similarly rapid (approximately 4 seconds), while in ATP-reduced and Taxol-treated cells, t(g) was increased to 210 seconds and 40 seconds, respectively. In detergent-extracted cells no recovery was seen. Consistent with the rapid dynamics of 5xGFP-EMTB measured with fluorescent speckle microscopy and FRAP, we estimated that the affinity of the MAP for microtubules is approximately 40 microM in untreated living cells, compared with approximately 1 microM in vitro. However, K(D,app) was not significantly changed in the presence of azide and was increased to 110 microM in the presence of Taxol. To test whether changes in the phosphorylation state of cellular proteins might be responsible for altering the dynamics of ensconsin binding, we used FSM to monitor staurosporine-treated cells. Staurosporine treatment substantially halted dynamics of 5xGFP-EMTB speckles along MTs. Our results show that ensconsin is highly dynamic in its association with microtubules, and its microtubule association can be altered by in vivo phosphorylation events.

This unit describes an in vitro assay that uses video-enhanced differential interference contrast (VE-DIC) microscopy to examine the motile interactions between isolated organelle fractions and microtubules (MTs). The method can be used to dissect the molecular requirements for organelle movement and membrane trafficking. A field of axoneme-nucleated MTs, growing and shortening as they would in a living cell (dynamic MTs), is generated in a simple microscope perfusion chamber. Various combinations of isolated endoplasmic reticulum (ER) and Golgi apparatus organelles, cytosol containing motor proteins and other soluble factors, nucleotides, and specific pharmacological reagents are then added to the dynamic MT, and the motile interactions between the organelles and MTs are observed by VE-DIC microscopy.

The GTPase Ran has recently been shown to stimulate microtubule polymerization in mitotic extracts, but its mode of action is not understood. Here we show that the mitotic role of Ran is largely mediated by the nuclear transport factor importin beta. Importin beta inhibits spindle formation in vitro and in vivo and sequesters an aster promoting activity (APA) that consists of multiple, independent factors. One component of APA is the microtubule-associated protein NuMA. NuMA and other APA components are discharged from importin beta by RanGTP and induce spindle-like structures in the absence of centrosomes, chromatin, or Ran. We propose that RanGTP functions in mitosis as in interphase by locally releasing cargoes from transport factors. In mitosis, this promotes spindle assembly by organizing microtubules in the vicinity of chromosomes.

To test how cell-cell contacts regulate microtubule (MT) and actin cytoskeletal dynamics, we examined dynamics in cells that were contacted on all sides with neighboring cells in an epithelial cell sheet that was undergoing migration as a wound-healing response. Dynamics were recorded using time-lapse digital fluorescence microscopy of microinjected, labeled tubulin and actin. In fully contacted cells, most MT plus ends were quiescent; exhibiting only brief excursions of growth and shortening and spending 87.4% of their time in pause. This contrasts MTs in the lamella of migrating cells at the noncontacted leading edge of the sheet in which MTs exhibit dynamic instability. In the contacted rear and side edges of these migrating cells, a majority of MTs were also quiescent, indicating that cell-cell contacts may locally regulate MT dynamics. Using photoactivation of fluorescence techniques to mark MTs, we found that MTs in fully contacted cells did not undergo retrograde flow toward the cell center, such as occurs at the leading edge of motile cells. Time-lapse fluorescent speckle microscopy of fluorescently labeled actin in fully contacted cells revealed that actin did not flow rearward as occurs in the leading edge lamella of migrating cells. To determine if MTs were required for the maintenance of cell-cell contacts, cells were treated with nocodazole to inhibit MTs. After 1-2 h in either 10 microM or 100 nM nocodazole, breakage of cell-cell contacts occurred, indicating that MT growth is required for maintenance of cell-cell contacts. Analysis of fixed cells indicated that during nocodazole treatment, actin became reduced in adherens junctions, and junction proteins alpha- and beta-catenin were lost from adherens junctions as cell-cell contacts were broken. These results indicate that a MT plus end capping protein is regulated by cell-cell contact, and in turn, that MT growth regulates the maintenance of adherens junctions contacts in epithelia.

Fluorescent speckle microscopy (FSM) is a new technique for visualizing the movement, assembly, and turnover of macromolecular assemblies like the cytoskeleton in living cells. In this method, contrast is created by coassembly of a small fraction of fluorescent subunits in a pool of unlabeled subunits. Random variation in association creates a nonuniform "fluorescent speckle" pattern. Fluorescent speckle movements in time-lapse recordings stand out to the eye and can be measured. Because fluorescent speckles represent fiduciary marks on the polymer lattice, FSM provides the opportunity for the first time to see the 2- and 3-dimensional trajectories of lattice movements within large arrays of polymers as well as identifying sites of assembly and disassembly of individual polymers. The technique works with either microinjection of fluorescently labeled subunits or expression of subunits ligated to green fluorescent protein (GFP). We have found for microtubules assembled in vitro that speckles containing one fluorophore can be detected and recorded using a conventional wide-field epi-fluorescence light microscope and digital imaging with a low noise cooled CCD camera. In living cells, optimal speckle contrast occurs at fractions of labeled tubulin of approximately 0.1-0.5% where the fluorescence of each speckle corresponds to one to seven fluorophores per resolvable unit (approximately 0.27 microm) in the microscope. This small fraction of labeled subunits significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells.

Microtubules are involved in actin-based protrusion at the leading-edge lamellipodia of migrating fibroblasts. Here we show that the growth of microtubules induced in fibroblasts by removal of the microtubule destabilizer nocodazole activates Rac1 GTPase, leading to the polymerization of actin in lamellipodial protrusions. Lamellipodial protrusions are also activated by the rapid growth of a disorganized array of very short microtubules induced by the microtubule-stabilizing drug taxol. Thus, neither microtubule shortening nor long-range microtubule-based intracellular transport is required for activating protrusion. We suggest that the growth phase of microtubule dynamic instability at leading-edge lamellipodia locally activates Rac1 to drive actin polymerization and lamellipodial protrusion required for cell migration.

The migration of tissue cells requires interplay between the microtubule and actin cytoskeletal systems. Recent reports suggest that interactions of microtubules with actin dynamics creates a polarization of microtubule assembly behavior in cells, such that microtubule growth occurs at the leading edge and microtubule shortening occurs at the cell body and rear. Microtubule growth and shortening may activate Rac1 and RhoA signaling, respectively, to control actin dynamics. Thus, an actin-dependent gradient in microtubule dynamic-instability parameters in cells may feed back through the activation of specific signalling pathways to perpetuate the polarized actin-assembly dynamics required for cell motility.

Fluorescence microscopic visualization of fluorophore-conjugated proteins that have been microinjected or expressed in living cells and have incorporated into cellular structures has yielded much information about protein localization and dynamics [1]. This approach has, however, been limited by high background fluorescence and the difficulty of detecting movement of fluorescent structures because of uniform labeling. These problems have been partially alleviated by the use of more cumbersome methods such as three-dimensional confocal microscopy, laser photobleaching and photoactivation of fluorescence [2]. We report here a method called fluorescent speckle microscopy (FSM) that uses a very low concentration of fluorescent subunits, conventional wide-field fluorescence light microscopy and digital imaging with a low-noise, cooled charged coupled device (CCD) camera. A unique feature of this method is that it reveals the assembly dynamics, movement and turnover of protein assemblies throughout the image field of view at diffraction-limited resolution. We found that FSM also significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells. Our initial applications include the measurement of microtubule movements in mitotic spindles and actin retrograde flow in migrating cells.

The dynamics of microtubules in living cells can be seen by fluorescence microscopy when fluorescently labeled tubulin is microinjected into cells, mixing with the cellular tubulin pool and incorporating into microtubules. The subsequent fluorescence distribution along microtubules can appear "speckled" in high-resolution images obtained with a cooled CCD camera (Waterman-Storer and Salmon, 1997. J. Cell Biol. 139:417-434). In this paper we investigate the origins of these fluorescent speckles. In vivo microtubules exhibited a random pattern of speckles for different microtubules and different regions of an individual microtubule. The speckle pattern changed only after microtubule shortening and regrowth. Microtubules assembled from mixtures of labeled and unlabeled pure tubulin in vitro also exhibited fluorescent speckles, demonstrating that cellular factors or organelles do not contribute to the speckle pattern. Speckle contrast (measured as the standard deviation of fluorescence intensity along the microtubule divided by the mean fluorescence intensity) decreased as the fraction of labeled tubulin increased, and it was not altered by the binding of purified brain microtubule-associated proteins. Computer simulation of microtubule assembly with labeled and unlabeled tubulin showed that the speckle patterns can be explained solely by the stochastic nature of tubulin dimer association with a growing end. Speckle patterns can provide fiduciary marks in the microtubule lattice for motility studies or can be used to determine the fraction of labeled tubulin microinjected into living cells.

Background: The microtubule-dependent motility of endoplasmic reticulum (ER) tubules is fundamental to the structure and function of the ER. From in vitro assays, three mechanisms for ER tubule motility have arisen: the 'membrane sliding mechanism' in which ER tubules slide along microtubules using microtubule motor activity; the 'microtubule movement mechanism' in which ER attaches to moving microtubules; and the 'tip attachment complex (TAC) mechanism' in which ER tubules attach to growing plus ends of microtubules., Results: We have used multi-wavelength time-lapse epifluorescence microscopy to image the dynamic interactions between microtubules (by microinjection of X-rhodamine-labeled tubulin) and ER (by DiOC6(3) staining) in living cells to determine which mechanism contributes to the formation and motility of ER tubules in migrating cells in vivo. Newly forming ER tubules extended only in a microtubule plus-end direction towards the cell periphery: 31.4% by TACs and 68.6% by the membrane sliding mechanism. ER tubules, statically attached to microtubules, moved towards the cell center with microtubules through actomyosin-based retrograde flow. TACs did not change microtubule growth and shortening velocities, but reduced transitions between these states. Treatment of cells with 100 nM nocodazole to inhibit plus-end microtubule dynamics demonstrated that TAC motility required microtubule assembly dynamics, whereas membrane sliding and retrograde-flow-driven ER motility did not., Conclusions: Both plus-end-directed membrane sliding and TAC mechanisms make significant contributions to the motility of ER towards the periphery of living cells, whereas ER removal from the lamella is powered by actomyosin-based retrograde flow of microtubules with ER attached as cargo. TACs in the ER modulate plus-end microtubule dynamics.

Fast axonal transport is characterized by the bidirectional, microtubule-based movement of membranous organelles. Cytoplasmic dynein is necessary but not sufficient for retrograde transport directed from the synapse to the cell body. Dynactin is a heteromultimeric protein complex, enriched in neurons, that binds to both microtubules and cytoplasmic dynein. To determine whether dynactin is required for retrograde axonal transport, we examined the effects of anti-dynactin antibodies on organelle transport in extruded axoplasm. Treatment of axoplasm with antibodies to the p150(Glued) subunit of dynactin resulted in a significant decrease in the velocity of microtubule-based organelle transport, with many organelles bound along microtubules. We examined the molecular mechanism of the observed inhibition of motility, and we demonstrated that antibodies to p150(Glued) disrupted the binding of cytoplasmic dynein to dynactin and also inhibited the association of cytoplasmic dynein with organelles. In contrast, the anti-p150(Glued) antibodies had no effect on the binding of dynactin to microtubules nor on cytoplasmic dynein-driven microtubule gliding. These results indicate that the interaction between cytoplasmic dynein and the dynactin complex is required for the axonal transport of membrane-bound vesicles and support the hypothesis that dynactin may function as a link between the organelle, the microtubule, and cytoplasmic dynein during vesicle transport.

We have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus ends of lamella MTs persist in growth perpendicular to the leading edge until they reach the base of the lamellipodium, where they oscillate between short phases of growth and shortening. Occasionally "pioneering" MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow. MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge. Both parallel MTs and photoactivated fluorescent marks on perpendicular MTs move rearward at the 0.4 mircon/min rate of retrograde flow in the lamella. MT rearward transport persists when MT dynamic instability is inhibited by 100-nM nocodazole but is blocked by inhibition of actomyosin by cytochalasin D or 2,3-butanedione-2-monoxime. Rearward flow appears to cause MT buckling and breaking in the lamella. 80% of free minus ends produced by breakage are stable; the others shorten and pause, leading to MT treadmilling. Free minus ends of unknown origin also depolymerize into the field of view at the lamella. Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that approximately 80% of the MTs in the lamella are not centrosome bound. We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.

Although it is generally believed that microtubules have minus ends bound to the centrosome and free plus ends that exhibit dynamic instability, recent observations show that the minus ends can be free and that modulation of dynamic instability at both ends can result in treadmilling and flux in interphase cells.

The distribution and dynamics of the membranous organelles in two cell types were investigated during cell division. Live cells (either PtK2 or LLC-PK1) labeled with the vital dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] were observed via serial optical sectioning with the laser-scanning confocal microscope. Z-series of labeled, dividing cells were collected every 1-2 minutes throughout mitosis, beginning at prophase and extending to the spreading of the daughter cells. Membrane distribution began to change from the onset of prophase in both cell types. When the mitotic spindle formed in prometaphase, fine tubular membranes, similar to those extending out to the edges of interphase cells aligned along the kinetochore spindle fibers. The lacy polygonal network typical of interphase cells persisted beneath the spindle, and a membrane network was also associated with the dorsal layer of the cell. As PtK2 cells reached metaphase, their spindles were nearly devoid of membrane staining, whereas the spindles of LLC-PK1 cells contained many tubular and small vesicular membranous structures. X-Z series of the LLC-PK1 metaphase spindle revealed a small cone of membranes that was separated from the rest of the cytoplasm by kinetochore MTs. In both cell types, as chromosome separation proceeded, the interzone remained nearly devoid of membranes until the onset of anaphase B. At this time the elongating interzonal microtubules were closely associated with the polygonal network of endoplasmic reticulum. Cytokinesis caused a compression, and then an exclusion of organelles from the midbody. Immunofluorescence staining with anti-tubulin antibodies suggested that spindle membranes were associated with microtubules throughout mitosis. In addition, taxol induced a dense and extensive collection of small vesicles to collect at the spindle poles of both cell types. Nocodazole treatment induced a distinct loss of organization of the membranous components of the spindles. Together these results suggest that microtubules organize the membrane distribution in mitotic cells, and that this organization may vary in different cell types depending on the quantity of microtubules within the spindle.

The myofibrillar cytoskeleton of skeletal muscle is made up of two distinct sets of filaments, the exosarcomeric cytoskeleton and the endosarcomeric cytoskeleton. The exosarcomeric cytoskeleton consists of intermediate filaments (IF) composed of the proteins desmin, vimentin, and synemin. The IF are arranged both longitudinally and transversely around the fiber. The longitudinal filaments run from Z-disc to Z-disc, enveloping the myofibril in order to serve as attachment sites for mitochondria, nuclei, and the sarcolemma, as well as limiting the sarcomere's extensibility. The transverse filaments link adjacent myofibrils at the Z-disc and are responsible for the fibril's axial register, and thus the striated appearance of muscle. The endosarcomeric cytoskeleton acts as a third filament system that coexists with actin and myosin within the sarcomere. This system is believed to be extensible and is made up of the giant proteins, titin and nebulin. Titin is believed to be responsible for resting muscle elasticity, as well as the central position of myosin in the sarcomere. Nebulin's role is proposed to be the maintenance of actin's lattice array. Following various types of intense exercise, pathological changes in muscle morphology have been documented. These include Z-disc streaming, sarcomerogenesis, and decentralization of myosin filaments within the sarcomere. It is hypothesized that disruption of the transverse IF system may cause Z-disc streaming, whereas degradation of titin filaments may affect myosin's position in the sarcomere.