A transgenic toolkit for visualizing and perturbing microtubules reveals unexpected functions in the epidermis
- Duke University Medical Center, United States
Figures
Figure 1 with 2 supplements
TRE-EB1 mouse line permits visualization of microtubule dynamics in vivo.
(A) Diagram of the TRE-EB1-GFP transgene. (B) Cross-section of e17.5 CMV-rtTA; TRE-EB1 epidermis. Scale-20μm. (C) Representative standard deviation projections of a basal, spinous, and granular …
Figure 1—figure supplement 1
Validation of EB1-GFP line and characterization of microtubule density.
(A) EB1-GFP labeling of a mitotic spindle at sub-cellular resolution in a basal keratinocyte. Scale-5µm. (B) Quantification of EB1-GFP density in indicated cell types. n = 25 cells for each cell …
Figure 1—figure supplement 2
Correlation analysis of microtubule parameters for individual microtubules in the indicated cell types.
Note the loss of correlation between growth distance and duration in granular cells.
Figure 2
TRE-spastin expression perturbs microtubules in vitro and in vivo.
(A) Diagram of the TRE-spastin transgene. (B) Spastin OE perturbs microtubules in cultured cells. Scale-10µm. (C) Insets from (B) showing microtubule density within individual cells based on spastin …
Figure 3
Spastin OE in basal keratinocytes induces mitotic arrest but does not alter epidermal architecture.
(A) Alleles used to induce spastin overexpression in basal keratinocytes. (B) HA-spastin expression in e16.5 embryonic epidermis. Scale-25µm. (C) Spastin OE causes microtubule loss, assayed using …
Figure 4 with 5 supplements
Spastin OE in differentiated keratinocytes induces cell-shape changes and entosis.
(A) Alleles used to overexpress spastin in suprabasal keratinocytes. (B) Images and quantification of HA-spastin expression in control and K10-rtTA; TRE-spastin epidermis. Asterisks indicate …
Figure 4—figure supplement 1
K10-rtTA expression faithfully recapitulates endogenous K10 expression.
(A) Alleles and experimental scheme used to validate K10-rtTA expression. (B) K10-rtTA induction begins at e14.and is uniform by e15.5. Scale-25μm. (C) Examples of robust K10-rtTA induction in …
Figure 4—figure supplement 2
Characterization of K10-rtTA; TRE-spastin epidermis.
(A) P0 control and K10-rtTA; TRE-spastin mice. (B) Cleaved caspase-3 staining in control and K10-rtTA; TRE-spastin epidermis. (C) Quantification of cleaved-caspase-3-positive cells in control and …
Figure 4—figure supplement 3
Granular cells fail to flatten in the K14-rtTA; TRE-spastin epidermis.
(A) HA-spastin cells in the K14-rtTA; TRE-spastin epidermis fail to flatten, in contrast to their wild-type neighbors. Asterisk indicates autofluorescence from cornified envelope. Scale-25µm. Zoomed …
Figure 4—figure supplement 4
Localization of E-cadherin in control and K10-rtTA; TRE-spastin epidermis.
Scale-25µm.
Figure 4—figure supplement 5
Spastin OE in suprabasal keratinocytes in adult mice perturbs epidermal homeostasis.
(A) Cross-section of adult (P45) epidermis in control and K10-rtTA; TRE-spastin mice demonstrating epidermal thickening in the mutant. (B) Spastin OE in adult mice causes a thickening of the …
Figure 5 with 1 supplement
Non-cell-autonomous desmosome defects in K10-rtTA; TRE-spastin epidermis.
(A) Immunofluorescence of desmosome components in control and K10-rtTA; TRE-spastin epidermis. Scale-25µm. (B,C) Quantifications of desmoplakin and DSC2/3 immunofluorescence at cell-cell boundaries …
Figure 5—figure supplement 1
Characterization of desmosomes in K14-rtTA; TRE-spastin epidermis.
(A) Desmoplakin immunofluorescence in P0 control and K14-rtTA; TRE-spastin epidermis with a high number of spastin-expressing suprabasal cells. (B) Quantification of desmoplakin immunofluorescence …
Figure 6 with 1 supplement
Proper corneocyte formation requires microtubules, but microtubule loss does not impair epidermal barrier function.
(A) CE is thickened in K10-rtTA; TRE-spastin tissue. Red lines indicate CE thickness. Scale-10µm. (B) Transmission electron micrographs of cornified envelopes in control and K10-rtTA; TRE-spastin …
Figure 6—figure supplement 1
Spastin OE does not impair tight junction localization or function.
(A) ZO-1 localization in control and K10-rtTA; TRE-spastin epidermis. Scale-25µm. (B) Region where spastin-positive cells are next to spastin-negative cells in K10-rtTA; TRE-spastin tissue. Note …
Videos
Video 1
EB1-GFP dynamics in a proliferative, basal keratinocyte in a mouse embryo.
https://doi.org/10.7554/eLife.29834.002
Video 2
EB1-GFP dynamics in a differentiated, spinous keratinocyte in a mouse embryo.
https://doi.org/10.7554/eLife.29834.003
Video 3
EB1-GFP dynamics in a differentiated, granular keratinocyte in a mouse embryo.
https://doi.org/10.7554/eLife.29834.004
Video 4
Z-stack of an example of entosis in K10-rtTA; TRE-spastin epidermis.
Phalloidin marks the cell outlines.
Tables
Table 1
Quantifications of microtubule parameters in indicated cell types.
Data are represented as mean ± standard deviation. n = 160 microtubules for each cell type.
| EB1 density (puncta/100 µm2) | Mean growth distance (µm) | Mean growth speed (µm/min) | Mean growth duration (s) | ||
|---|---|---|---|---|---|
| In vivo | Basal | 9.1 ± 2.1 | 3.1 ± 1.77 | 11.09 ± 3.11 | 17.84 ± 10.98 |
| Spinous | 8.68 ± 2.3 | 2.1 ± 1.5 | 12.2 ± 3.27 | 10.24 ± 6.32 | |
| Granular | 17.39 ± 4.64 | 0.89 ± 0.57 | 7.1 ± 3.66 | 8.49 ± 4.33 | |
| Primary | Basal | 3.56 ± 1.17 | 6.74 ± 3.9 | 29.23 ± 9.19 | 14.63 ± 9.11 |
| Suprabasal | 4.23 ± 1.76 | 5.31 ± 3.34 | 31.46 ± 7.97 | 10.15 ± 5.62 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.29834.022
