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Alpha actinin-1 regulates cell-matrix adhesion organization in keratinocytes: consequences for skin cell motility.

Hamill KJ, Hiroyasu S, Colburn ZT, Ventrella RV, Hopkinson SB, Skalli O, Jones JC - J. Invest. Dermatol. (2014)

Bottom Line: Keratinocytes deficient in ACTN1 exhibit changes in their actin cytoskeleton organization, a loss in front-rear polarity, and impaired lamellipodial dynamics.They also display aberrant directed motility and move slower compared with their wild-type counterparts.In the same cells, hemidesmosome proteins arrange in cat paw patterns, more typical of confluent, stationary cells, and β4 integrin dynamics are reduced in knockdown cells compared with control keratinocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Eye and Vision Science, Institute of Ageing and Chronic Diseases, University of Liverpool, Liverpool, UK.

ABSTRACT
The migration of keratinocytes in wound healing requires coordinated activities of the motility machinery of a cell, the cytoskeleton, and matrix adhesions. In this study, we assessed the role of alpha actinin-1 (ACTN1), one of the two alpha actinin isoforms expressed in keratinocytes, in skin cell migration via a small hairpin RNA-mediated knockdown approach. Keratinocytes deficient in ACTN1 exhibit changes in their actin cytoskeleton organization, a loss in front-rear polarity, and impaired lamellipodial dynamics. They also display aberrant directed motility and move slower compared with their wild-type counterparts. Moreover, they have abnormally arranged matrix adhesion sites. Specifically, the focal adhesions in ACTN1 knockdown keratinocytes are not organized as distinct entities. Rather, focal adhesion proteins are arranged in a circle subjacent to cortical fibers of actin. In the same cells, hemidesmosome proteins arrange in cat paw patterns, more typical of confluent, stationary cells, and β4 integrin dynamics are reduced in knockdown cells compared with control keratinocytes. In summary, our data suggest a mechanism by which ACTN1 determines the motility of keratinocytes by regulating the organization of the actin cytoskeleton, focal adhesion, and hemidesmosome proteins complexes, thereby modulating cell speed, lamellipodial dynamics, and directed migration.

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ACTN1 knockdown impacts lamellipodial dynamics(a) Representative phase-contrast images of iHEKs, iHEKs expressing scrambled shRNA and the three ACTN1 knockdown clones plated overnight on glass bottomed dishes. (b) Mean ± s.e. cell body and lamellipodial area determined from images from 3 independent experiments, 50–100 cells/group. (c) Cells were scored based on the number of lamellipodial protrusions and plotted as percentage of the population displaying 0, 1, 2, or 3+ lamellipodia. (d–g) Phase contrast images of cells were captured every 5s over 10mins and kymographs generated as a montage of the pixels beneath a line drawn in the direction of the largest lamellipodial protrusion. (d) Representative kymographs from each cell line with time on the vertical axis. Example measurement sites of extension persistence (time spent in elongation phase) and extension distance (length of extension from base of previous retraction event) are indicated. Mean ± s.e plots of extension persistence (e), extension distance (f) and extension rate (g). Plots are derived from 25–50 cell/line in three independent studies. Bars in a and d, 10 µm. In c, e and f, * denotes significant differences from iHEK and scrambled shRNA controls groups as determined by ANOVA, p<0.05.
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Figure 4: ACTN1 knockdown impacts lamellipodial dynamics(a) Representative phase-contrast images of iHEKs, iHEKs expressing scrambled shRNA and the three ACTN1 knockdown clones plated overnight on glass bottomed dishes. (b) Mean ± s.e. cell body and lamellipodial area determined from images from 3 independent experiments, 50–100 cells/group. (c) Cells were scored based on the number of lamellipodial protrusions and plotted as percentage of the population displaying 0, 1, 2, or 3+ lamellipodia. (d–g) Phase contrast images of cells were captured every 5s over 10mins and kymographs generated as a montage of the pixels beneath a line drawn in the direction of the largest lamellipodial protrusion. (d) Representative kymographs from each cell line with time on the vertical axis. Example measurement sites of extension persistence (time spent in elongation phase) and extension distance (length of extension from base of previous retraction event) are indicated. Mean ± s.e plots of extension persistence (e), extension distance (f) and extension rate (g). Plots are derived from 25–50 cell/line in three independent studies. Bars in a and d, 10 µm. In c, e and f, * denotes significant differences from iHEK and scrambled shRNA controls groups as determined by ANOVA, p<0.05.

Mentions: As mentioned above, our immunofluorescence analyses suggest that ACTN1 knockdown cells display polarity defects. To investigate this further, images of live individual cells plated overnight on glass-bottomed dishes were captured and cell surface area, lamellipodial area and number of lamellipodial protrusions were determined (Figure 4a). Although ACTN1 knockdown keratinocytes occasionally display slightly smaller cell body area than parental iHEK, the difference from controls is below significance (Figure 4b). In addition, their lamellipodial area, a combination of the area covered by their small multiple cell surface extensions, remains unchanged (Figure 4b). However, there is a significant decrease in ACTN1-knockdown lines exhibiting a single lamellipodium in comparison to control iHEKs (Figure 4c). This confirms that knockdown cells show a reduction in intrinsic frontrear polarity.


Alpha actinin-1 regulates cell-matrix adhesion organization in keratinocytes: consequences for skin cell motility.

Hamill KJ, Hiroyasu S, Colburn ZT, Ventrella RV, Hopkinson SB, Skalli O, Jones JC - J. Invest. Dermatol. (2014)

ACTN1 knockdown impacts lamellipodial dynamics(a) Representative phase-contrast images of iHEKs, iHEKs expressing scrambled shRNA and the three ACTN1 knockdown clones plated overnight on glass bottomed dishes. (b) Mean ± s.e. cell body and lamellipodial area determined from images from 3 independent experiments, 50–100 cells/group. (c) Cells were scored based on the number of lamellipodial protrusions and plotted as percentage of the population displaying 0, 1, 2, or 3+ lamellipodia. (d–g) Phase contrast images of cells were captured every 5s over 10mins and kymographs generated as a montage of the pixels beneath a line drawn in the direction of the largest lamellipodial protrusion. (d) Representative kymographs from each cell line with time on the vertical axis. Example measurement sites of extension persistence (time spent in elongation phase) and extension distance (length of extension from base of previous retraction event) are indicated. Mean ± s.e plots of extension persistence (e), extension distance (f) and extension rate (g). Plots are derived from 25–50 cell/line in three independent studies. Bars in a and d, 10 µm. In c, e and f, * denotes significant differences from iHEK and scrambled shRNA controls groups as determined by ANOVA, p<0.05.
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Figure 4: ACTN1 knockdown impacts lamellipodial dynamics(a) Representative phase-contrast images of iHEKs, iHEKs expressing scrambled shRNA and the three ACTN1 knockdown clones plated overnight on glass bottomed dishes. (b) Mean ± s.e. cell body and lamellipodial area determined from images from 3 independent experiments, 50–100 cells/group. (c) Cells were scored based on the number of lamellipodial protrusions and plotted as percentage of the population displaying 0, 1, 2, or 3+ lamellipodia. (d–g) Phase contrast images of cells were captured every 5s over 10mins and kymographs generated as a montage of the pixels beneath a line drawn in the direction of the largest lamellipodial protrusion. (d) Representative kymographs from each cell line with time on the vertical axis. Example measurement sites of extension persistence (time spent in elongation phase) and extension distance (length of extension from base of previous retraction event) are indicated. Mean ± s.e plots of extension persistence (e), extension distance (f) and extension rate (g). Plots are derived from 25–50 cell/line in three independent studies. Bars in a and d, 10 µm. In c, e and f, * denotes significant differences from iHEK and scrambled shRNA controls groups as determined by ANOVA, p<0.05.
Mentions: As mentioned above, our immunofluorescence analyses suggest that ACTN1 knockdown cells display polarity defects. To investigate this further, images of live individual cells plated overnight on glass-bottomed dishes were captured and cell surface area, lamellipodial area and number of lamellipodial protrusions were determined (Figure 4a). Although ACTN1 knockdown keratinocytes occasionally display slightly smaller cell body area than parental iHEK, the difference from controls is below significance (Figure 4b). In addition, their lamellipodial area, a combination of the area covered by their small multiple cell surface extensions, remains unchanged (Figure 4b). However, there is a significant decrease in ACTN1-knockdown lines exhibiting a single lamellipodium in comparison to control iHEKs (Figure 4c). This confirms that knockdown cells show a reduction in intrinsic frontrear polarity.

Bottom Line: Keratinocytes deficient in ACTN1 exhibit changes in their actin cytoskeleton organization, a loss in front-rear polarity, and impaired lamellipodial dynamics.They also display aberrant directed motility and move slower compared with their wild-type counterparts.In the same cells, hemidesmosome proteins arrange in cat paw patterns, more typical of confluent, stationary cells, and β4 integrin dynamics are reduced in knockdown cells compared with control keratinocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Eye and Vision Science, Institute of Ageing and Chronic Diseases, University of Liverpool, Liverpool, UK.

ABSTRACT
The migration of keratinocytes in wound healing requires coordinated activities of the motility machinery of a cell, the cytoskeleton, and matrix adhesions. In this study, we assessed the role of alpha actinin-1 (ACTN1), one of the two alpha actinin isoforms expressed in keratinocytes, in skin cell migration via a small hairpin RNA-mediated knockdown approach. Keratinocytes deficient in ACTN1 exhibit changes in their actin cytoskeleton organization, a loss in front-rear polarity, and impaired lamellipodial dynamics. They also display aberrant directed motility and move slower compared with their wild-type counterparts. Moreover, they have abnormally arranged matrix adhesion sites. Specifically, the focal adhesions in ACTN1 knockdown keratinocytes are not organized as distinct entities. Rather, focal adhesion proteins are arranged in a circle subjacent to cortical fibers of actin. In the same cells, hemidesmosome proteins arrange in cat paw patterns, more typical of confluent, stationary cells, and β4 integrin dynamics are reduced in knockdown cells compared with control keratinocytes. In summary, our data suggest a mechanism by which ACTN1 determines the motility of keratinocytes by regulating the organization of the actin cytoskeleton, focal adhesion, and hemidesmosome proteins complexes, thereby modulating cell speed, lamellipodial dynamics, and directed migration.

Show MeSH
Related in: MedlinePlus