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Brown CM, Hebert B, Kolin DL, Zareno J, Whitmore L, Horwitz AR, Wiseman PW (2006) Probing the integrin-actin linkage using high-resolution protein velocity mapping. J. Cell Science. 119(Pt 24): 5204-14. Abstract (Pubmed)

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Cell migration is regulated in part by the connection between the substratum and the actin cytoskeleton. However, the very large number of proteins involved in this linkage and their complex network of interactions make it difficult to assess their role in cell migration. We apply a novel image analysis tool, spatio-temporal image correlation spectroscopy (STICS), to quantify the directed movements of adhesion-related proteins and actin in protrusions of migrating cells. The STICS technique reveals protein dynamics even when protein densities are very low or very high, and works in the presence of large, static molecular complexes. Detailed protein velocity maps for actin and the adhesion-related proteins alpha-actinin, alpha5-integrin, talin, paxillin, vinculin and focal adhesion kinase are presented. The data show that there are differences in the efficiency of the linkage between integrin and actin among different cell types and on the same cell type grown on different substrate densities. We identify potential mechanisms that regulate efficiency of the linkage, or clutch, and identify two likely points of disconnect, one at the integrin and the other at alpha-actinin or actin. The data suggests that the efficiency of the linkage increases as actin and adhesions become more organized showing the importance of factors that regulate the efficiency in adhesion signaling and dynamics.
 

Carbonell WS, Murase S, Horwitz AF, Mandell JW (2005) Migration of perilesional microglia after focal brain injury and modulation by CC chemokine receptor 5: An in situ time-lapse confocal imaging study. Link to PubMed

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Microglia rapidly become reactive in response to diverse stimuli and are thought to be prominent participants in the pathophysiology of both acute injury and chronic neurological diseases. However, mature microglial reactions to a focal lesion have not been characterized dynamically in adult vertebrate tissue. Here, we present a detailed analysis of long-distance perilesional microglial migration using time-lapse confocal microscopy in acutely isolated living slices from adult brain-injured mice. Extensive migration of perilesional microglia was apparent by 24 h after injury and peaked at 3 d. Average instantaneous migration speeds of approximately 5 microm/min and peak speeds >10 microm/min were observed. Collective, directed migration toward the lesion edge was not observed as might be expected in the presence of chemoattractive gradients. Rather, migration was autonomous and could be modeled as a random walk. Pharmacological blockade of the cysteine-cysteine chemokine receptor 5 reduced migration velocity and the number of perilesional migratory microglia without affecting directional persistence, suggesting a novel role for chemokines in modulation of discrete migratory parameters. Finally, activated microglia in the denervated hippocampal stratum oriens did not migrate extensively, whereas human immunodeficiency virus-1 tat-activated microglia migrated nearly twice as fast as those at the stab lesion, indicating a nonuniform microglial response to different stimuli. Understanding the characteristics and specific molecular mechanisms underlying microglial migration after neural injury could reveal novel targets for therapeutic strategies for modulating neuroinflammation in human diseases.
   
Wiseman PW, Brown CM, Webb DJ, Herbert B, Johnson NL, Squier JA, Ellisman MH, Horwitz AF (2004) Spatial mapping of integrin interactions and dynamics during cell migration by image correlation microscopy. Journal of Cell Science. 117(Pt 23): 5521-34. Abstract (PubMed)

Image Correlation Microscopy (ICM) was used to characterize alpha5 integrin clustering, diffusion and interactions in the cell. ICM allows the detection of submicroscopic alpha5 integrin clusters with 3-4 proteins. A temporal ICM analysis reveals heterogenity in both alpha5 integrin and alpha-actinin dynamics across the cell with the proteins being more dynamic in regions of the cell that are ruffling and protruding. Two color ICM shows that alpha5 integrin and alpha-actinin localize and move together even in regions of the cell with no discernable adhesions. When adhesions disassemble three proteins have very different fates: alpha5 integrin diffuses away slowly, alpha-actinin moves away with a directed motion and paxillin diffuses away rapidly into the cytosol.
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Supplemental Movies

( from Publications)

Carbonell WS, Murase S, Horwitz AF, and Mandell JW (2005) Infiltrative microgliosis: activation and long-distance migration of subependymal microglia following periventricular insults. Journal of Neuroinflamation. 2(1): 5. Abstract (PubMed)

SVM phagocytosis of ependymal debris. Activity of SVMs suggestive of phagocytosis of dye-labeled ependymal cell debris 24 h following injection. SVMs can be seen extending processes towards debris. Examples of ependymal debris are pseudocolored yellow. Each frame is a 2D projection representing a stack of 6 images 8 um apart. Original magnification, 40x.

Dynamics of infiltrative microgliosis. Infiltrative microgliosis of SVMs into the hippocampul stratum oriens from the subependymal region of the posterior lateral ventricle. Note highly directed migration into the hippocampus. Each frame is a 2D projection representing a stack of 4 images 10 um apart taken every 3 minutes. Original magnification, 20x.

 

Webb DJ, Donais K, Whitmore LA, Thomas SM, Turner CE, Parsons JT, Horwitz AF (2004) FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion diassembly. Nat Cell Biol. 6(2): 154-161. Abstract (PubMed)

Time-lapse images for Figure 1 showing an MEF expressing paxillin-GFP. min. Paxillin localized in adhesions near the leading edge of the lamellipodium and the adhesions at the base of protrusions disappeared as new adhesions formed; this is termed adhesion turnover. Actual duration is 60

Time-lapse images for Figure 1, which shows an MEF expressing paxillin-DsRed2. Actual duration is 60 min.

 

Murase S and Horwitz AF (2002) Deleted in Colorectal Carcinoma and Differentially Expressed Integrins Mediate the Directional Migration of Neural Precursors in the Rostral Migratory Stream. Journal of Neuroscience. 22(9): 3568-3579. Abstract (PubMed)

Three neural precursors migrating to the olfactory bulb (top) at the center of the panel, and is the movie from which Figure 5A (Murase & Horwitz, 2002) is derived. The overall direction is toward the olfactory bulb, even though temporal retrograde moving of a cell body occurs (see Figure 5B). The slice for this experiment is from P12 mouse. Actual duration of the movie is 100 minutes, and is composed of 20 frames (5 min intervals between each frame).

Movie shows the inhibitory effect of anti-integrin alphav antibody on the migration of neural precursors, and corresponds to Figure 5C (Murase & Horwitz, 2002). The olfactory bulb is located at the right of the panel. The slice for this experiment is from a P12 mouse. The cell membrane undulates rapidly, but the cell itself does not move. Actual duration of the movie is 100 minutes, and composed of 20 frames (5 min intervals between each frame).

Shows the bizarre protrusion of the leading process of migrating cells under the treated with an anti-DCC Mab. The unidirectional movement of the leading process observed in Movie 1 is not seen in these cells. The slice is from a P3 mouse, and the tendency to meander without clear direction of the cells suggests that DCC plays a role of direction finding sensor. The movie corresponds to Figure 7E (Murase & Horwitz, 2002), and composed of 42 frames with 5 min intervals.  Actual duration of the movie is 210 min.

This time-lapse movie shows cells moving at the border between the end portion of the rostral migratory stream (RMS) and granule cell layer; that is, these cells are beginning to differentiate to mature neurons.  The rapid movement of cells in the RMS is not found, but cells extend multi-branched leading processes without clear direction. The slice is from a P2 mouse, and the movie is composed of 153 frames with 5 min intervals. Actual duration is 765 min.

 

Images & Movies

(low resolution movies for easy downloading)
Dynamics of the "molecular feet" of a migrating connective tissue cell. (5.6 MB) (D. Webb, K. Donais)
GFP_paxillin confocal movie

MEF cell transfected with GFP-Paxillin, DIC image overlay. (C. Brown)

Hippocampal Neuron alpha-actinin-GFP

Hippocampal neuron transfected with alpha-actinin-GFP. (H.Zhang)

Neuronal Process  movie

Elongation of neuronal process in hippocampal neuron. (D.Webb, H. Asmussen)

Beta-1 integrin in DRG neurons

Endogenous beta-1 integrin localizes to the tips of growth cones in DRG neurons isolated from day 10 chick embryos, plated on FN and immunostained for beta-1 integrin.(D.Webb, H. Asmussen)

Hippocampal neuron expressing alpha-actinin-GFP movie

Hippocampal neurons expressing alpha-actinin-GFP. (D.Webb, H. Asmussen)

Hippocampal neuron stained with Synapsin & MAP2

Hippocampal neuron in culture (green-Synapsin1, red-MAP2). (H.Zhang)

DiI neural precurser cells

DiI labelled neural precursor cells migrating toward the olfactory bulb (left). (S.Murase)

Hippocampal Neuron movie

Hippocampal neuron transfected with beta-actin-GFP. (H.Zhang)

Granule cell migration movie Tangentially migrating granule cells in the pre-migratory zone in the postnatal cerebellum.GFP-labelled cells with long preocesses are migrating bi-directionally. (S.Murase)
FRAP

CHOB-2 cell stably transfected with alph5-GFP. An area of the cell was traced for pattern photobleaching. (C. Brown)

Muscle precursor cell migration in vivo movie

Migration of muscle precursor cells from the somite to forelimb. (B. Knight)

GFP_Paxillin_DIC Overlay

GFP-Paxillin and DIC image overlay. (C. Brown)

CHOB2-GFP-alpha5 movie

CHOB-2 cell stably transfected with GFP-alpha5. (D. Webb, K. Donais)

Map4 mitosis picture

Map4-mitosis in CHOK-1 cells (L.Whitmore, C. Brown)

CHOK!-GFP-alpha-actinin movie

CHOK-1 cell stably transfected with GFP-alpha-actinin. (D. Webb, K. Donais)

WI38 cell migration movie

Migration of a WI38 cell transfected with alpha-5-GFP. Notice the "tracks" left behind on the substrate. (D.Webb, K. Donais)

CHOK1-GFP-Paxillin movie

CHOK-1 cell stably transfected with GFP-paxillin. Notice the "turnover" of adhesions in the front of the cell. (D.Webb, K. Donais)

GFP-AKT-PH movie

GFP-AKT-PH in migrating neurons (chicken cerebellar tissue culture).(A. Sakakibara)

Map4 confocal movie

Map4 (C. Brown)


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