Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons
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During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored.
We show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia are motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin.
We propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.
KeywordsGolgi Apparatus Primary Cilium Migrate Neuron Fluorescence Recovery After Photobleaching Medial Ganglionic Eminence
Cerebellar granule neuron
Fluorescence recovery after photobleaching
Medial ganglionic eminence
Red fluorescent protein
Teal fluorescent protein
Utrophin actin-binding domain
Yellow fluorescent protein.
The necessity of neuronal migration for appropriate nervous system lamination and circuit formation has spurred intense investigation into the molecular and cellular mechanisms of this crucial morphogenic movement [1, 2, 3]. In most brain regions, neurons use a stereotypical saltatory motility cycle involving a sequential organelle transport and adhesion/de-adhesion events to migrate along their substrates [4, 5, 6, 7, 8, 9, 10]. They first elaborate a leading process that adheres to substrates (e.g., glial cells, axons) and guides the direction of migration. Next, in some populations of migrating neurons, including cerebellar granule neurons (CGNs), pyramidal neurons, and gonadotropin-releasing hormone neurons, an F-actin- and myosin ii motor-enriched region of the leading process proximal to the neuronal soma [11, 12, 13, 14, 15] (sometimes called the cytoplasmic dilation ) becomes engorged with cytoplasmic components, including the centrosome and Golgi apparatus [17, 18, 19, 20, 21, 22, 23, 24]. After the centrosome translocates through the leading process, the nucleus follows and the sequence is repeated until the neuron reaches its destined cortical lamina. The significance of this two-stroke sequence is illustrated by its conservation in neurons throughout the vertebrate brain and by its apparent requirement for appropriate migration, as perturbation of cytoskeletal and signaling components essential for migration strongly affect the choreography of the motility cycle [13, 15, 19, 21, 22, 23, 25].
The two-stroke nucleokinesis cycle has served as the main model for studies of the polarity of migrating neurons and the spatiotemporal roles of cytoskeletal components in migration. While disturbance of the microtubule and actin cytoskeletons is known to perturb the two-stroke cycle, only recently have time-lapse imaging studies provided mechanistic insight into the coordination of the motility cycle. Cytoplasmic dynein motors are localized at the base of the neuron’s leading process and soma, where they are thought to generate pulling forces on microtubules that help position the centrosome and facilitate subsequent somal translocation . The leading process is also a site of F-actin dynamics accumulation and myosin ii motor activity [11, 12, 13, 15]. Myosin ii-powered actin flow in the direction of migration is required for centrosome positioning and eventual somal translocation.
Despite these advances, it has remained unexplored whether the cytoskeletal forces that position the centrosome are unique to this organelle or apply more broadly to other events linked to two-stroke motility cycle and ultimately to the polarity of migrating neurons. We were curious whether leading-process actomyosin cytoskeleton coordinates the positioning of other cytoplasmic organelles, how organelle positioning is coordinated with substrate adhesion, and whether actomyosin organizes the overall polarity of migrating neurons. We generated a panel of time-lapse imaging probes to examine, for the first time, the dynamic distribution of the Golgi apparatus, primary cilia, and cell-cell adhesions in cultured CGNs migrating along glial fibers – a well-established model for radial neuronal migration. We used time-lapse imaging to mechanistically test the hypothesis that leading-process actomyosin controls both global organelle positioning and the loci of adhesive traction in the leading process. We show that the motility of the Golgi apparatus, which has been postulated via examination of fixed neurons to undergo two-stroke movement, depends on myosin ii motor activity. Further, the polarized transport of the primary cilia (consistent with the two-stroke cycle) requires myosin ii motors and F-actin cytoskeletal dynamics. Finally, we found that the formation and turnover of adhesions in the F-actin-enriched region of the leading process and soma requires the actomyosin cytoskeleton. Imaging of adhesion dynamics revealed that the neuronal soma appears to slide past adhesions before their final disassembly in the CGN trailing process, suggesting adhesion disassembly is not precisely linked to somal advance. Taken together, these findings demonstrate that the leading-process actomyosin traction forces not only position multiple cytoplasmic organelles but also regulate adhesion formation and turnover that precedes somal translocation. Considering that asymmetric organelle transport and cell-cell adhesion distributions are hallmarks of cell polarity, we propose that myosin ii no longer be viewed only as a motor that powers cytoskeletal arrangement for neuronal migration; instead, we propose that it be viewed as broadly coordinating the overall polarity of migrating neurons.
Results and discussion
The Golgi apparatus displays two-stroke motility that parallels F-actin dynamics
Myosin ii and F-actin dynamics are required for Golgi apparatus translocation in migrating CGNs
Myosin ii and F-actin dynamics are required for basal motility of the Golgi apparatus in stationary CGNs
Translocation of primary cilia displays two-stroke motility and parallels F-actin dynamics
To expand our examination of organelle positioning during the first phase of the two-stroke cycle, we next assessed the motility of primary cilia in migrating CGNs. While mother centriole positioning and primary cilia have been examined in migrating interneurons [31, 32], we found no reports describing a direct examination of the motility of primary cilia during radial migration. The primary cilium, composed of a membrane-docked mother centriole, axonemal microtubules, and a cilia-associated plasma membrane [33, 34, 35], differs from the Centrin2-labeled daughter centriole examined in previous imaging studies [11, 18, 19, 21, 22, 23], which is primarily a cytoplasmic organelle with limited attachment to cytoskeletal or membrane structures . The primary cilium provides an opportunity to examine the interaction of the actomyosin cytoskeleton with the positioning of discrete membrane and cytoskeletal domains.
Myosin ii and F-actin dynamics are required for translocation of primary cilia in migrating CGNs
Myosin ii and F-actin dynamics are required for basal motility of primary cilia in stationary CGNs
Jam-C and Cadm3 cell-cell contacts are enriched in the leading process base and soma near actin accumulation sites
Although studies have begun to examine the location of adhesive contacts and trafficking of adhesion receptors in migrating neurons (reviewed in [39, 40]), a significant remaining challenge is to unravel the relationship between the sites of cell contact and the underlying cytoskeletal elements that control adhesion in migrating neurons. Recent findings in non-neuronal cell motility suggest that myosin ii motors and the actin cytoskeleton recruited to sites of cell contact play a direct role in strengthening substrate adhesion in the leading portion of migrating cells and that tension generated by myosin ii-based adhesion forward of the nucleus is required for efficient cell motility [41, 42, 43, 44]. Given the expanding known functions of leading-process actomyosin in neurons [11, 12, 13, 15] and the role of actomyosin in controlling adhesion in non-neuronal cells, we next tested whether leading-process actomyosin regulates adhesion dynamics in migrating neurons.
We examined the localization of pHluorin-tagged Cadm3, an immunoglobulin superfamily adhesion molecule expressed in CGNs at neuron-neuron and neuron-glial contacts , to determine whether the observed dynamics of JAM-C were unique or applied to other adhesion molecules. Time-lapse imaging of CGNs expressing Cadm3 pHluorin and either UTRCH-ABD-EGFP or Lifeact-EGFP revealed surface Cadm3 at punctate junctions within the leading process and in larger adhesion plaques within the neuronal soma (Figure 8C,D, Movies in Additional files 28, 29, 30 and 31). Adaptive volumetric kymographs and analysis of the percentage of Cadm3-pHluorin fluorescence in the leading-, trailing-process, or soma revealed the Cadm3-pHluorin-labeled puncta also appeared in F-actin-enriched regions of the proximal leading process and soma. Similar to JAM-C, Cadm3-pHluorin levels were initially high in the leading process and then coalesced into the trailing process as the cell body translocated past adhesions that were initially located in the leading process. Taken together these results suggest that the main sites of JAM-C and Cadm3 adhesive cell contact reside within the soma and the proximal portion of the leading process in migrating CGNs, near regions of F-actin enrichment.
Myosin ii and F-actin dynamics are required for adhesion dynamics in migrating CGNs
Studies in our laboratory previously showed that leading-process actomyosin flow orchestrates the saltatory movement of CGNs along glial fibers by controlling positioning of the centrosome and soma during the two-stroke nucleokinesis cycle . However, the overall function of actomyosin contractility in relation to the polarity of the two-stroke motility cycle displayed by migrating neurons remains to be elucidated. Here, we have shown that leading-process actomyosin globally coordinates the positional polarity of multiple organelles before nucleokinesis and regulates adhesive traction forces within the leading process of the migrating neuron. We would like to note that, while the preponderance of actomyosin in the CGN leading process suggests this is the main site for actin and myosin coordination of these events, we cannot discount that minor populations of actin in other regions of the cell may also contribute until more focal techniques are applied to CGNs.
The orientation of the Golgi apparatus defines the axis of polarity and the vectorial flow of membrane traffic in a variety of cell types. The Golgi apparatus reorients toward the “leading edge” in epithelial or astrocyte monolayers induced to migrate in scratch assays [50, 51], reorients toward immune synapses in immune cells [52, 53], and localizes to axons or dendrites at various stages of neuronal polarization [54, 55, 56, 57]. Golgi polarization during neuronal migration was first observed in ultrastructural studies [58, 59] and later confirmed in inhibitory rat cortical interneurons  and tangentially migrating mouse MGE neurons by using specific Golgi markers . Using fixed MGE cultures, Bellion et al. showed the Golgi to be located within the dilated region of the MGE leading process before nuclear translocation , a finding recently confirmed by in vivo time lapse imaging . However, the two-stroke motility of this organelle has not been examined in radially migrating neurons and the cytoskeletal components that control its motility have not been identified. Time-lapse imaging of CGNs shows that not only does the Golgi enter the leading process during the first stroke of the nucleokinesis cycle but it also moves with timing and velocity similar to that of the centrosome.
We also showed that F-actin is enriched in the CGN leading process when the Golgi participates in the first step of the two-stroke nucleokinesis cycle, suggesting that the leading-process actomyosin flow responsible for CGN centrosome positioning plays a role in Golgi motility. Direct manipulation of the actomyosin cytoskeleton by blebbistatin and jasplakinolide treatment revealed that myosin ii motor activity and F-actin dynamics are required for Golgi positioning before nuclear translocation. This result was surprising, as actin-based motors like myosin ii  and myosin vi  have been shown only to play roles in Golgi membrane fusion and fission, not positioning. In most models, cytoplasmic dynein is thought to be the predominant motor regulating Golgi positioning by directing Golgi membranes to the vicinity of the centrosome . Interestingly, Zmuda and Rivas reported that an intact actin cytoskeleton is required for re-alignment of the Golgi within the CGN cell body as CGNs initiate parallel fibers axons prior to migration . Thus, the actin cytoskeleton and myosin ii motors are likely to play an important role in the positioning of the Golgi apparatus, a role that is distinct from the centrosome-directing activity of cytoplasmic dynein.
Primary cilia have recently been reported to transduce crucial signaling, for example, sonic hedgehog and sdf-1-based signaling, that regulates neuron migration in the developing cerebral cortex [31, 32]. Interestingly, preliminary imaging studies have described the primary cilia as motile in cortical interneurons. The dynamics of primary cilia have also not been examined in radially migrating neurons, nor have the cytoskeletal components required for the motility of this organelle been reported in any cell type. In migrating CGNs, primary cilia are localized largely in the proximal portion of the leading process; they move toward the future direction of migration during the first step of the two-stroke nucleokinesis cycle. Imaging of the F-actin cytoskeleton and myosin ii motors demonstrated that the actomyosin cytoskeleton accumulates with the primary cilia during the two-stroke cycle. Myosin ii motor activity and F-actin dynamics are also required for motility of the primary cilia, as blebbistatin and jasplakinolide block the movement of cilia in both migrating and stationary neurons. These results expand the role of the actin cytoskeleton in regulating aspects of cilium biology (reviewed in [34, 63]).
A recent functional genomics screen identified a number of actin regulatory proteins that control the length of primary cilia . While the role of the actin cytoskeleton in regulating the positioning of primary cilia has not been studied in any system, there is compelling evidence that actomyosin regulates the positioning of motile cilia without affecting their beating. Myosin ii motors and actin biochemically fractionate with purified basal body preparations from heavily ciliated cells  and appear to accumulate at the base of motile cilia in ultrastructural localization studies [65, 66], much as we observed for CGN primary cilia. Moreover, basal bodies associate with actomyosin before they migrate to the apical domain of polarized epithelial cells , and treatment with drugs that inhibit myosin ii motors prevents the docking of the basal body with the cortical actin cytoskeleton [67, 68]. Our findings suggest that the actomyosin association with primary cilia may represent a conserved feature of primary and motile cilia that orients their direction of motility during neuron migration.
Recent advances have revealed a diversity of mechanisms that regulate the adhesion of neurons to their migration substrates; these include adhesion receptor endocytosis, Reelin regulation of pro-adhesive small GTPases, phosphorylation of GAP junctional proteins, and ubiquitin proteasome degradation of polarity proteins [47, 69, 70, 71, 72, 73, 74, 75]. Despite these advances, there remains much to be learned about how adhesion dynamics in the two-stroke motility cycle are controlled in live, migrating neurons. Most studies have examined adhesion receptor localization in fixed cells or have not specifically examined cell surface adhesion receptors. Our pHluorin-based imaging probes allowed us to directly examine the cell surface adhesion dynamics at CGN-Bergmann glial junctions in migrating CGNs. JAM-C and Cadm3 formed punctate junctions in the base of the leading process and larger plaques in the soma, as previously described in ultrastructural studies [58, 59] and more recent studies of the localization of astrotactin1 and β1 integrin adhesion receptors [69, 72]. In our study, before somal movement, adhesions formed within actin-rich regions at the base of the leading process and then coalesced into larger plaques within the soma. Previous models of adhesion disassembly during neuronal migration proposed that nucleokinesis and somal translocation would occur simultaneously with adhesion disassembly at the rear of the soma of a migrating neuron [69, 72]. Surprisingly, we observed the soma to slide past many adhesions before these finally disassembled in the CGN trailing process, suggesting that traction forces generated by adhesions within the leading process are more important for somal translocation than their disassembly in the trailing aspect of the neuron. Finally, the actomyosin cytoskeleton in the vicinity of CGN junctions appears to regulate adhesion dynamics, as inhibition of myosin ii motors and slowed F-actin dynamics rendered JAM-C and Cadm3 adhesions static and appeared to block their assembly, disassembly, and movement within the plasma membrane (a finding confirmed and quantified by FRAP). While no previous study has linked the actomyosin cytoskeleton to control of adhesion dynamics in migrating neurons, these results are consistent with recent advances in general models of cell motility. In fibroblasts and epithelial cells, myosin ii bundling of actin filaments and actin polymerization controls adhesion morphology [41, 42, 43, 44, 76]. In these systems, nascent adhesions form within actin-rich regions of lamellipodia mature as myosin ii bundles actin filaments within the lamella just forward of the nucleus and disassemble due to actomyosin activity in the rear of the cell. Our results, together with previous findings about leading-process actomyosin flow [11, 12], suggest that the base of the leading process is similar to the lamella of epithelial cells and fibroblasts, as all are actomyosin-rich regions forward of the nucleus that support adhesion dynamics necessary for efficient cell migration.
How does actomyosin coordinate the events that are linked to initial steps of the two-stroke motility cycle prior to nuclear movement? The work of our lab and others shows not only that actomyosin accumulates in the leading process of migrating neurons but also that it flows down the leading process in a myosin ii-dependent fashion. Moreover, specific inactivation of leading-process actomyosin motors halts neuronal migration . It is likely that either direct or indirect linkage between the cortical actomyosin cytoskeleton and organelles or adhesion molecules are necessary to harness actomyosin flow and drive the organelle or surface receptor movements that occur during migration (Additional file 36: Figure S4). When considering actomyosin flow, the movement of the primary cilia and adhesion receptors likely represent the most local activity of the actomyosin cytoskeleton as they occur within the plane of the cell membrane closest to the actomyosin rich cortex. In the future, it will be of great interest to identify the molecules that link organelle movement to actomyosin flow in migrating neurons.
Taken together, our findings show that asymmetric organelle positioning, membrane organization, and adhesion formation define the polarity of nucleokinesis in radially migrating neurons, much as in classical cell polarity models (e.g., epithelial apicobasal polarity or front-to-back polarity in migrating fibroblasts). In polarized epithelia and fibroblasts, myosin ii motor activity executes cell polarity programs after polarity is initiated [77, 78]. Similarly, our results demonstrate that myosin ii activity is required to power many key events linked to polarity during nucleokinesis. Overall, we propose that myosin ii no longer be viewed only as a motor that powers cytoskeletal arrangement for migration; instead, we propose that it be viewed as broadly coordinating the overall polarity of the migrating neuron. In the future, it will be of great interest to determine how extrinsic factors that instruct leading-process extension and ultimately the direction of migration contribute to the asymmetric recruitment of actomyosin flow necessary for polarized organelle positioning and adhesion formation. It will also be of interest to determine whether key polarity proteins, such as the partitioning defective complex, regulate Golgi or cilia positioning and leading process adhesive traction through an interaction with myosin ii motors.
All mice in this study were housed, bred, and used according to procedures and guidelines approved by Institutional Animal Care and Use Committee at St. Jude Children’s Research Hospital (protocol number = 483).
Preparation and nucleofection of CGNs
CGNs were prepared as previously described (Hatten, 1985). Briefly, cerebella were dissected from the brains of P7 C57BL6 mice, the pia was peeled away, and cerebellar tissue was treated with trypsin and triturated using a fine-bore fire-polished Pasteur pipettes. A single-cell suspension in CMF-PBS was layered onto a 60% to 35% Percoll gradient, and separated by centrifugation. The cellular fraction at the interphase was isolated (routinely contained 95% CGNs and 5% glia) and transfected by nucleofection. For imaging experiments, CGNs were nucleofected with 1 to 20 μg of pCIG2 expression vector encoding fluorescence-labeled cytoskeletal proteins by using the Amaxa Mouse Neuron kit with A030 or O-005 program on the Amaxa Nucleofector II system (Lonza). Nucleofected CGNs were plated in glass-bottomed movie dishes (Mattek) coated with poly-L-ornithine and Matrigel at a low concentration to facilitate attachment of neurons to glial processes.
All cDNAs encoding fluorescent fusion proteins were subcloned into the pCIG2 expression vector . pCIG2 H2B-mCherry and pCIG2 JAM-C-pHluorin have been previously described . Franck Polleux provided pCIG2 Lifeact-RFP and pCIG2 Lifeact-EGFP. Graham Warren provided the GalNAcT2-YFP cDNA. Robert Adelstein provided the MHCiiB cDNA. The Arl13b-Venus, Arl13b-KO2, PACT-KO, Cadm3-pHluorin, TFP-UTRCH-ABD, and TFP-Lifeact cDNAs were commercially synthesized and subcloned into pCIG2 by Genscript (Piscataway, NJ, USA).
Drug treatment experiments
For myosin ii inhibition or F-actin stabilization experiments, cells were imaged before drug addition, the indicated amount of blebbistatin or jasplakinolide was added to the bath, and the cultures were then imaged for an additional time. For acute migration experiments, migrating cells were imaged for 24 minutes (at 2 minute intervals), the indicated drugs were added to the bath, and the cultures were imaged for a further 36 minutes (total length of movie – 60 min, but time points 26 to 32 minutes were not included in analysis to avoid images that were out of focus or movement of cells caused by drug addition). For measurement of basal organelle movement, cells were imaged for 5 minutes at 15-second intervals. Cytoskeletal drugs were then added to the bath and movement was imaged every 10 minutes over the next hour using the same imaging conditions (5 minute movies with 15-second intervals) to examine the changes in organelle motility over time due to drug addition. All blebbistatin-sensitive organelle positioning events were also examined using Kusibira orange labeled imaging probes (e.g., Arl13, Centrin2, and GalNacT2) that were illuminated with 560 nm wavelength light to confirm reductions in organelle velocities were not due to phototoxicity sometimes associated with imaging of blebbistatin with intense blue wavelength light (data not shown). Blebbistatin is frequently used at concentrations between 25 to 100 μM (all of which have been tested on migrating CGNs) and even at 50 to 100 μM concentrations it is specific for inhibiting the related skeletal muscle and non-muscle myosin motors without affecting smooth muscle myosin, Myo 1b, MyoVa, or MyoX .
Imaging and analysis
CGN cultures were imaged with a Marianas Spinning Disk Confocal Microscope (Intelligent Imaging Innovations) comprising a Zeiss Axio Observer microscope equipped with 40×/1.0 NA (oil immersion) and 63×/1.4 NA (oil immersion) PlanApochromat objectives. An Ultraview CSUX1 confocal head with 440 to 514 nm or 488/561 nm excitation filters and ImageEM-intensified CCD camera (Hamamatsu) were used for high-resolution imaging. Z-stacks (10 μm thick, 13 sections per stack) were collected over the duration and time-intervals indicated. Movies were captured, processed, and analyzed using Slidebook software (Intelligent Imaging innovations). Generally, all image analysis was obtained using the manual tracking protocol tool in Slidebook to measure the average velocities of the organelles. Cells were only included into the study if they showed good health throughout the length of the movie, and the organelles of interest were visible over at least five consecutive frames. In acute drug treatment movies, neurons that moved more than two cell body lengths were considered to be migrating neurons.
Adaptive volumetric kymographs
Kymographs for each time sequence were generated by accumulating the fluorescence of the cell within its 3D segmentation boundaries. We refer to these as “volumetric kymographs” to differentiate them from the typical kymographs that are generated from 2D images. Assuming an accurate 3D segmentation of the cell, this approach provides a more accurate estimate of the fluorescent content of the cell because it takes into account the shape of the cell in the z-dimension, as opposed to summing or computing the max over all slices at each x-y location.
The 3D cell segmentation for a given cell was computed by manually selecting an intensity threshold for that cell and keeping only those voxels that are connected to the soma. In many cases, segmentation “walls” were necessary to prevent the segmentation from bleeding into an adjacent cell in the image. These walls acted as lines of zero intensity in the volume and were drawn manually in the sequence using a custom software program wherever the intensity-based segmentation captured part of a neighboring cell.
Once the 3D segmentation was computed, the volumetric kymograph was computed by analyzing cross-sections of the segmented cell perpendicular to a predefined vector. This vector was defined as the direction from the soma to the tip of the leading process in the first frame of the sequence, which approximates the cell’s direction of migration. Cross-sections of the cell were computed at a distance of one x-y pixel apart along the vector. Examples of these cross-sections can be seen in Supplemental Movies 4 and 5, where five such cross-sections are shown for illustration (but many more were computed). The volumetric kymograph was then generated by computing either the sum or the max of the pixel fluorescent intensities in each cross-section, resulting in a fluorescence signal along the length of the cell. Examples of these signals for the red and green channels are also shown in Supplemental Movies 4 and 5, plotted above the vector line.
Mean concentration analysis
For image sequences containing Golgi or cilia, the mean concentration of actin was computed in the vicinity of these objects. This was done by first marking the position of the object of interest at each time-point in the sequence and then defining a cylindrical region around the object of a desired radius, where the axis of the cylinder was perpendicular to the z-axis. The intersection of the segmented cell volume and the cylindrical volume was used as the region in which to accumulate actin concentration around the object, and the mean of the actin fluorescent intensity in that region was computed at each time-point. The mean of the actin fluorescence over the entire 3D segmented cell region was also computed as a baseline for comparison. Radii of 1, 2, and 4 μm were used in the computation to assess the actin concentration around the object at different scales.
FRAP analysis of migrating CGNs
CGNs were nucleofected for 18 hours with expression vectors encoding Jam-C-pHluorin or Cadm3-pHluorin and RFP-UTRCH-ABD. Cultures were then imaged with the Marianas confocal microscope described above, equipped with a Vector high-speed x,y scanner (Intelligent Imaging Innovations) fitted to emit 488-nm light for photobleaching pHluorin-labeled adhesion receptors. Regions of interest were photobleached using 30% laser power and a 20 ms dwell time, and images were acquired at 2-second intervals after bleaching. The half-life (t1/2) of Jam-C-pHluorin or Cadm3-pHluorin and mobile cell fraction were calculated using Slidebook.
We thank Atsushi Miyawaki for sharing the Venus cDNA, Franck Polleux for providing the Lifeact constructs, Graham Warren for providing GalNAcT2-YFP and Robert Adelstein for providing the MCHiiB cDNA. Sharon Naron provided expert editorial support. The Solecki Laboratory is funded by the American Lebanese Syrian Associated Charities (ALSAC), by grant #1-FY12-455 from the March of Dimes, and by grant 1R01NS066936 from the National Institute of Neurological Disorders (NINDS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NINDS or the NIH. The authors declare that they have no conflict of interest.
Additional file 25: Movie 22: Two-dimensional intensity plot of the imaging sequence shown in Figure 8A. The neuron in this sequence was segmented with the custom-designed algorithm described in Materials and Methods. A line approximating the vector of migration is shown in grey and selected orthogonal planes of the segmentation are shown in black. The fluorescence intensity of RFP-UTRCH (red) and JAM-C-pHluorin (green) along the extent of the entire neuron are displayed as a curve above the approximate region of the segmented cell. The fluorescence intensities of f-actin and JAM-C adhesions are highly coincident in the leading process and cell body, indicating that JAM-C adhesions are localized to f-actin rich regions of migrating neurons. (MOV 14 MB)
Additional file 27: Movie 24: Two-dimensional intensity plot of the imaging sequence shown in Figure 8B. The neuron in this sequence was segmented with the custom-designed algorithm described in Materials and Methods. A line approximating the vector of migration is shown in grey and selected orthogonal planes of the segmentation are shown in black. The fluorescence intensity of RFP-Lifeact (red) and JAM-C-pHluorin (green) along the extent of the entire neuron are displayed as a curve above the approximate region of the segmented cell. The fluorescence intensities of f-actin and JAM-C adhesions are highly coincident in the leading process and cell body, indicating that JAM-C adhesions are localized to f-actin-rich regions of migrating neurons. (MOV 15 MB)
Additional file 29: Movie 26: Two-dimensional intensity plot of the imaging sequence shown in Figure 8C. The neuron in this sequence was segmented with the custom-designed algorithm described in Materials and Methods. A line approximating the vector of migration is shown in grey and selected orthogonal planes of the segmentation are shown in black. The fluorescence intensity of RFP-UTRCH (red) and Cadm3-pHluorin (green) along the extent of the entire neuron are displayed as a curve above the approximate region of the segmented cell. The fluorescence intensities of f-actin and Cadm3 adhesions are highly coincident in the leading process and cell body, indicating that Cadm3 adhesions are localized to f-actin rich regions of migrating neurons. (MOV 12 MB)
Additional file 31: Movie 28: Two-dimensional intensity plot of the imaging sequence shown in Figure 8D. The neuron in this sequence was segmented with the custom-designed algorithm described in Materials and Methods. A line approximating the vector of migration is shown in grey and selected orthogonal planes of the segmentation are shown in black. The fluorescence intensity of RFP-Lifeact (red) and Cadm3-pHluorin (green) along the extent of the entire neuron are displayed as a curve above the approximate region of the segmented cell. The fluorescence intensities of f-actin Cadm3 adhesions are highly coincident in the leading process and cell body, indicating that Cadm3 adhesions are localized to f-actin-rich regions of migrating neurons. (MOV 9 MB)
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