scholarly journals Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells

2002 ◽  
Vol 158 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Wendy C. Salmon ◽  
Michael C. Adams ◽  
Clare M. Waterman-Storer
Keyword(s):  
Cell Body ◽  
Time Lapse ◽  
Lung Epithelial Cells ◽  
Dual Wavelength ◽  
Convergence Zone ◽  
Retrograde Flow ◽  
Filamentous Actin ◽  
Actin Bundles ◽  
Migrating Cells

Interactions between microtubules (MTs) and filamentous actin (f-actin) are involved in directed cell locomotion, but are poorly understood. To test the hypothesis that MTs and f-actin associate with one another and affect each other's organization and dynamics, we performed time-lapse dual-wavelength spinning-disk confocal fluorescent speckle microscopy (FSM) of MTs and f-actin in migrating newt lung epithelial cells. F-actin exhibited four zones of dynamic behavior: rapid retrograde flow in the lamellipodium, slow retrograde flow in the lamellum, anterograde flow in the cell body, and no movement in the convergence zone between the lamellum and cell body. Speckle analysis showed that MTs moved at the same trajectory and velocity as f-actin in the cell body and lamellum, but not in the lamellipodium or convergence zone. MTs grew along f-actin bundles, and quiescent MT ends moved in association with f-actin bundles. These results show that the movement and organization of f-actin has a profound effect on the dynamic organization of MTs in migrating cells, and suggest that MTs and f-actin bind to one another in vivo.

scholarly journals Regulation of leading edge microtubule and actin dynamics downstream of Rac1

2003 ◽  
Vol 161 (5) ◽  
pp. 845-851 ◽  
Author(s):  
Torsten Wittmann ◽  
Gary M. Bokoch ◽  
Clare M. Waterman-Storer
Keyword(s):  
Rho Gtpases ◽  
Actin Polymerization ◽  
Leading Edge ◽  
Time Lapse ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Retrograde Flow ◽  
Rho Proteins ◽  
Migrating Cells

Actin in migrating cells is regulated by Rho GTPases. However, Rho proteins might also affect microtubules (MTs). Here, we used time-lapse microscopy of PtK1 cells to examine MT regulation downstream of Rac1. In these cells, “pioneer” MTs growing into leading-edge protrusions exhibited a decreased catastrophe frequency and an increased time in growth as compared with MTs further from the leading edge. Constitutively active Rac1(Q61L) promoted pioneer behavior in most MTs, whereas dominant-negative Rac1(T17N) eliminated pioneer MTs, indicating that Rac1 is a regulator of MT dynamics in vivo. Rac1(Q61L) also enhanced MT turnover through stimulation of MT retrograde flow and breakage. Inhibition of p21-activated kinases (Paks), downstream effectors of Rac1, inhibited Rac1(Q61L)-induced MT growth and retrograde flow. In addition, Rac1(Q61L) promoted lamellipodial actin polymerization and Pak-dependent retrograde flow. Together, these results indicate coordinated regulation of the two cytoskeletal systems in the leading edge of migrating cells.

scholarly journals Actomyosin-based Retrograde Flow of Microtubules in the Lamella of Migrating Epithelial Cells Influences Microtubule Dynamic Instability and Turnover and Is Associated with Microtubule Breakage and Treadmilling

1997 ◽  
Vol 139 (2) ◽  
pp. 417-434 ◽  
Author(s):  
Clare M. Waterman-Storer ◽  
E.D. Salmon
Keyword(s):  
Epithelial Cells ◽  
Dynamic Instability ◽  
Leading Edge ◽  
Unknown Origin ◽  
Time Lapse ◽  
Lung Epithelial Cells ◽  
Retrograde Flow ◽  
Microtubule Dynamic ◽  
Lung Epithelial ◽  
Time Lapse Imaging

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 μm/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 ∼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.

scholarly journals Analysis of the Actin–Myosin II System in Fish Epidermal Keratocytes: Mechanism of Cell Body Translocation

1997 ◽  
Vol 139 (2) ◽  
pp. 397-415 ◽  
Author(s):  
Tatyana M. Svitkina ◽  
Alexander B. Verkhovsky ◽  
Kyle M. McQuade ◽  
Gary G. Borisy
Keyword(s):  
Cell Body ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Leading Edge ◽  
Time Lapse ◽  
Supramolecular Organization ◽  
Retrograde Flow ◽  
Body Boundary ◽  
Filament Bundles

While the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translocation, we analyzed the supramolecular organization of the actin–myosin II system and the dynamics of myosin II in fish epidermal keratocytes. In lamellipodia, long actin filaments formed dense networks with numerous free ends in a brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The polarity of actin filaments was almost uniform, with barbed ends forward throughout most of the lamellipodia but mixed in arc-shaped filament bundles at the lamellipodial/cell body boundary. Myosin II formed discrete clusters of bipolar minifilaments in lamellipodia that increased in size and density towards the cell body boundary and colocalized with actin in boundary bundles. Time-lapse observation demonstrated that myosin clusters appeared in the lamellipodia and remained stationary with respect to the substratum in locomoting cells, but they exhibited retrograde flow in cells tethered in epithelioid colonies. Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles. In locomoting cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propose that the forward translocation of the cell body and retrograde flow in the lamellipodia are both driven by contraction of an actin–myosin network in the lamellipodial/cell body transition zone.

scholarly journals The dynamic interplay between ATP/ADP levels and autophagy sustain neuronal migration in vivo

2020 ◽  
Author(s):  
Bressan Cedric ◽  
Pecora Alessandra ◽  
Gagnon Dave ◽  
Snapyan Marina ◽  
Labrecque Simon ◽  
...  
Keyword(s):  
Cell Migration ◽  
Stationary Phase ◽  
Neuronal Migration ◽  
Stationary Phases ◽  
Focal Adhesions ◽  
Time Lapse ◽  
List Type ◽  
Atp Levels ◽  
Migrating Cells

AbstractCell migration is a dynamic process that entails extensive protein synthesis and recycling, structural remodeling, and a considerable bioenergetic demand. Autophagy is one of the pathways that maintain cellular homeostasis. Time-lapse imaging of autophagosomes and ATP/ADP levels in migrating cells in the rostral migratory stream of mice revealed that decrease in ATP levels force cells into the stationary phase and induce autophagy. Genetic impairment of autophagy in neuroblasts using either inducible conditional mice or CRISPR/Cas9 gene editing decreased cell migration due to the longer duration of the stationary phase. Autophagy is modulated in response to migration-promoting and inhibiting molecular cues and is required for the recycling of focal adhesions. Our results show that autophagy and energy consumption act in concert in migrating cells to dynamically regulate the pace and periodicity of the migratory and stationary phases in order to sustain neuronal migration.HighlightsADP levels dynamically change during cell migrationA decrease in ATP levels leads to cell pausing and autophagy induction via AMPKAutophagy is required to sustain neuronal migration by recycling focal adhesionsAutophagy level is dynamically modulated by migration-promoting and inhibiting cues

FURTHER OBSERVATIONS ON AMOEBOID HAEMOCYTES IN BLABERUS GIGANTEUS (L.) (ORTHOPTERA: BLATTIDAE)

1961 ◽  
Vol 39 (5) ◽  
pp. 755-766 ◽  
Author(s):  
J. W. Arnold
Keyword(s):  
Cell Body ◽  
Time Lapse ◽  
Flat Surfaces ◽  
Variable Form ◽  
Form And Function ◽  
Projection Analysis ◽  
Rapid Elongation ◽  
Wing Veins ◽  
And Function

The movements of amoeboid haemocytes in vivo in wing veins of B. giganteus were studied with the aid of time-lapse cinephotomicrography and projection analysis. They are described here in detail and discussed in relation to haemocyte form and function. Haemocyte motion included non-migratory as well as migratory aspects. Non-migratory motion comprised the slow to turbulent cytoplasmic motion and intermittent probing movements of stationary cells. Active migration occurred in the more or less typical amoeboid fashion and also in the more peculiar contractile manner which involved the projection of hyaline, tactile pseudopodia of variable form and often resulted in extreme and relatively rapid elongation of the cell body. Haemocytes were thus able to move on flat surfaces, to extend themselves across spaces, and to force their way into narrow tissue interstices. These activities demonstrate the versatility of the cells and provide a means of accounting for certain of their functions.

scholarly journals Automated Multichamber Time-lapse Videography for Long-term In Vivo Observation of Migrating Cells

In Vivo ◽  
2017 ◽  
Vol 31 (3) ◽  
pp. 329-334 ◽  
Author(s):  
HELMUT BUHLER ◽  
RAPHAEL ADAMIETZ ◽  
THERESA ABELN ◽  
DAVID DIAZ-CARBALLO ◽  
PASCALINE NGUEMGO-KOUAM ◽  
...  
Keyword(s):  
Time Lapse ◽  
Migrating Cells

scholarly journals The dynamic interplay between ATP/ADP levels and autophagy sustain neuronal migration in vivo

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Cedric Bressan ◽  
Alessandra Pecora ◽  
Dave Gagnon ◽  
Marina Snapyan ◽  
Simon Labrecque ◽  
...  
Keyword(s):  
Cell Migration ◽  
Stationary Phase ◽  
Neuronal Migration ◽  
Stationary Phases ◽  
Focal Adhesions ◽  
Time Lapse ◽  
Time Lapse Imaging ◽  
Migratory Stream ◽  
Migrating Cells

Cell migration is a dynamic process that entails extensive protein synthesis and recycling, structural remodeling, and considerable bioenergetic demand. Autophagy is one of the pathways that maintain cellular homeostasis. Time-lapse imaging of autophagosomes and ATP/ADP levels in migrating cells in the rostral migratory stream of mouse revealed that decreases in ATP levels force cells into the stationary phase and induce autophagy. Pharmacological or genetic impairments of autophagy in neuroblasts using either bafilomycin, inducible conditional mice, or CRISPR/Cas9 gene editing decreased cell migration due to the longer duration of the stationary phase. Autophagy is modulated in response to migration-promoting and inhibiting molecular cues and is required for the recycling of focal adhesions. Our results show that autophagy and energy consumption act in concert in migrating cells to dynamically regulate the pace and periodicity of the migratory and stationary phases to sustain neuronal migration.

scholarly journals Photoreceptor progenitor dynamics in the zebrafish embryo retina and its modulation by primary cilia and N-cadherin

2020 ◽  
Vol 52 ◽  
Author(s):  
Gonzalo Aparicio ◽  
Magela Rodao ◽  
José L. Badano ◽  
Flavio R. Zolessi
Keyword(s):  
Cell Body ◽  
Primary Cilia ◽  
Outer Nuclear Layer ◽  
Time Lapse ◽  
Apical Surface ◽  
Apical Position ◽  
Transgenic Expression ◽  
Basal Process ◽  
Maturation Stages

Photoreceptors of the vertebrate neural retina are originated from the neuroepithelium, and like other neurons, must undergo cell body translocation and polarity transitions to acquire their final functional morphology, which includes features of neuronal and epithelial cells. We analyzed this process in detail on zebrafish embryos using in vivo confocal microscopy and electron microscopy. Photoreceptor progenitors were labeled by the transgenic expression of EGFP under the regulation of the photoreceptor-specific promoter crx, and structures of interest were disrupted using morpholino oligomers to knock-down specific genes. Photoreceptor progenitors detached from the basal retina at pre-mitotic stages, rapidly retracting a short basal process as the cell body translocated apically. They remained at an apical position indefinitely to form the outer nuclear layer (ONL), initially extending and retracting highly dynamic neurite-like processes, tangential to the apical surface. Many photoreceptor progenitors presented a short apical primary cilium. The number and length of these cilia was gradually reduced until nearly disappearing around 60 hpf. Their disruption by knocking-down ift88 and elipsa caused a notorious defect on basal process retraction. To assess the role of cell adhesion in the organization of photoreceptor progenitors, we knocked-down cdh2/N-cadherin and observed the cell behavior by time-lapse microscopy. The ectopic photoreceptor progenitors initially migrated in an apparent random manner, profusely extending cell processes, until they encountered other cells to establish cell rosettes in which they stayed acquiring the photoreceptor-like polarity. Altogether, our observations indicate a complex regulation of photoreceptor progenitor dynamics to form the retinal ONL, previous to the post-mitotic maturation stages.

scholarly journals Growth Cone Collapse through Coincident Loss of Actin Bundles and Leading Edge Actin without Actin Depolymerization

2001 ◽  
Vol 153 (5) ◽  
pp. 1071-1084 ◽  
Author(s):  
Feng-quan Zhou ◽  
Christopher S. Cohan
Keyword(s):  
Growth Cone ◽  
Kinase Inhibitor ◽  
Leading Edge ◽  
Time Lapse ◽  
Growth Cones ◽  
Filamentous Actin ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Growth Cone Collapse ◽  
Substrate Adhesion

Repulsive guidance cues can either collapse the whole growth cone to arrest neurite outgrowth or cause asymmetric collapse leading to growth cone turning. How signals from repulsive cues are translated by growth cones into this morphological change through rearranging the cytoskeleton is unclear. We examined three factors that are able to induce the collapse of extending Helisoma growth cones in conditioned medium, including serotonin, myosin light chain kinase inhibitor, and phorbol ester. To study the cytoskeletal events contributing to collapse, we cultured Helisoma growth cones on polylysine in which lamellipodial collapse was prevented by substrate adhesion. We found that all three factors that induced collapse of extending growth cones also caused actin bundle loss in polylysine-attached growth cones without loss of actin meshwork. In addition, actin bundle loss correlated with specific filamentous actin redistribution away from the leading edge that is characteristic of repulsive factors. Finally, we provide direct evidence using time-lapse studies of extending growth cones that actin bundle loss paralleled collapse. Taken together, these results suggest that actin bundles could be a common cytoskeletal target of various collapsing factors, which may use different signaling pathways that converge to induce growth cone collapse.

Contractile Filopodia and in Vivo Cell Movement in the Tunic of the Ascidian, Botryllus Schlosseri

1974 ◽  
Vol 15 (3) ◽  
pp. 513-535
Author(s):  
C. S. IZZARD
Keyword(s):  
Cell Body ◽  
Significant Role ◽  
Cell Movement ◽  
Time Lapse ◽  
Botryllus Schlosseri ◽  
Change Direction ◽  
The Matrix

The in vivo movement of one class of cells in the tunic of the ascidian Botryllus schlosseri has been analysed using differential interference optics and time-lapse cinematography. Long (up to 200 µm), thin (0.35-0.5 µm diameter) filopodia radiate from the cell-body into the matrix of the tunic. Movement of the cell-body consists of a series of short, jerky displacements with frequent changes in direction between successive displacements. The net displacement of the cell may be extremely small when the displacements are short and frequently change direction, or considerable when successive displacements show a persistence of direction (up to 114 µm in 60 min). Deformation of the elastic cuticle covering the tunic at points of attachment of the filopodia has been used to record qualitatively changes in tension in the filopodia. Correlation of the changes in tension with changes in length of the filopodia and movement of the cell-body have permitted the following conclusions. Active contractions of filopodia (i.e. increase in tension during shortening) stretch and move the cell-body. These movements exert a force on trailing or opposing filopodia. Relaxations of filopodia (i.e. decrease in tension during lengthening) result in small movements of the cell-body due to the recoil of tension in the cell-body and opposing filopodia. The position of the cell-body in space at any one instant in time is therefore the resultant of the forces developed in all the filopodia. Movement results from unilateral modulation of the tension developed in the filopodia. Active contractions play a more significant role in movement than relaxations.

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