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 keratinocyte. Scale-10μm. (D) Kymographs of EB1-GFP in indicated cell types. Scale-1µm. (E) Quantification of microtubule growth distance. (F) Quantification of microtubule growth speed. (G) Quantification of duration of microtubule growth. n = 160 microtubules for each stage. Data are presented as mean ± S.E.M. n.s.-p>0.05, *p<0.05. ***p<0.001.
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 type. (C) Single frame of EB1-GFP in granular cells in e17.5 embryos treated with either DMSO or nocodazole. Scale-10µm. Data are represented as mean ± S.E.M. n.s.-p>0.05, ***p<0.001.
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 expression. Scale-2µm. (D) Quantification of microtubule perturbation following spastin OE. n = 50 cells each from two independent experiments. (E) Spastin expression was observed within 24 hr of doxycycline exposure and no leaky expression was detected in TRE-spastin mice. Scale-25µm. (F) Weights of CMV-rtTA; TRE-spastin mice and control littermates following doxycycline exposure. (G) HA-spastin expression in various tissues after 96 hr of doxycycline exposure. Scale-25µm. (H) Effects of spastin OE on microtubule density in vivo. Note that the cilia in the kidney are dramatically shortened. Scale for the heart and liver microtubules-10µm. Scale for the cilia-5µm.
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 the 3xGFP-ensconcin microtubule-binding domain (EMTB) mouse (Lechler and Fuchs, 2007), and mitotic arrest. Scale-10µm. (D) Arrows indicate mitotically arrested cells in K14-rtTA; TRE-spastin epidermis. Scale-25µm. (E) Quantification of the number of basal keratinocytes in mitosis. n = 3 mice per genotype. (F) Quantification of mitotic stage in control back skin and spastin-negative and spastin-positive cells in K14-rtTA; TRE-spastin back skin. n = 3 mice per genotype. (cell type x mitotic stage interaction, p<0.0001). (G) Quantification of cleaved-caspase-3-positive cells at the indicated stages. (H) Quantification of BrdU+ basal cells in control and mutant back skin at the indicated stages. (I) X-gal barrier assay in e18.5 embryos. (J) Expression of keratins 5/14 and keratin 1 in control and K14-rtTA; TRE-spastin epidermis. Scale-50µm. Insets show zoomed regions illustrating cells expressing both K5/14 and K1 (white arrows) and also suprabasal cells that are only K5/14+. Scale-10µm. (K) Delaminating mitotic cells in K14-rtTA; TRE-spastin epidermis. Scale-10µm. Data are presented as mean ± S.E.M. *p<0.05. **p<0.01.
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 autofluorescence of the cornified envelope. Scale-25µm. Quantification is the percentage of suprabasal cells expressing spastin averaged over 5 different mice. (C) Hematoxylin and eosin staining of control and K10-rtTA; TRE-spastin tissue. Note the cornified envelope thickness. Scale-25µm. (D) Quantification of epidermal thickness in control and K10-rtTA; TRE-spastin mice. Each column is 120 measurements from 4 mice per genotype. (E) Quantification of the number of cell layers present in control and K10-rtTA; TRE-spastin epidermis. n = 100 measurements from 4 mice per genotype. (F) Quantification of BrdU+ basal cells in control and K10-rtTA; TRE-spastin epidermis. n = 4 mice per genotype. (G) Cell rounding is observed in a cell-autonomous manner in K10-rtTA; TRE-spastin tissue. Scale-25μm. Zoomed regions show a spastin-negative and a spastin-positive cell within K10-rtTA; TRE-spastin tissue. Note the accompanying aspect ratios (AR). Scale-10μm. (H) Quantification of the aspect ratio of individual control and spastin-positive cells. n = 100 cells for each group. (I). Spastin-positive granular cells remain flattened after short spastin induction. Scale-10µm. (J) Isolated granular cells treated with DMSO or nocodazole. Scale-10µm. (K) Example of an entotic cell in K10-rtTA; TRE-spastin epidermis. Scale-25μm. (L) Quantification of the number of entotics per mm of basement membrane. n = 4 mice per genotype. (M) Example of an entosis where the invading cell has up-regulated phospho-myosin light chain II. The dotted line marks the cell outlines. Scale-10μm. (N) Example of cell potentially invading its neighbor. Scale-10µm. (O) Examples of types of entosis observed in K10-rtTA; TRE-spastin epidermis. Scale-10μm. Data are presented as mean ± S.E.M. *p<0.05, ***p<0.001.
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 multiple tissues in P0 pups. Scale (backskin, top)−200 μm. Scale (paw, bottom left)−200 μm. Scale (back skin, tongue, palate, tail, bottom right)−25 μm. (D) Examples of robust K10-rtTA induction across multiple tissues in adult (P30) mice. Note that in the tail, where endogenous K10 is restricted to the interscale region, H2B-GFP expression is only observed in interscale regions. Scale-25μm.
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 K10-rtTA; TRE-spastin epidermis. n = 4 mice for each genotype. (D) Epidermal cross-sections stained for markers of stress (K6), stratification (K5/14 and K10), and terminal differentiation (loricrin and filaggrin). All scale bars are 25 μm. Data are presented as mean ± S.E.M. n.s.-p>0.05.
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 regions with associated aspect ratios for outlined cells are shown below. Scale-10µm. (B) Quantification of the aspect ratio of individual control and spastin-positive cells. n > 100 cells for each group. *p<0.05, ***p<0.001.
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 suprabasal, K10-positive layers of the interfollicular epidermis. Dashed lines indicate the basement membrane. All scale bars are 10 µm.
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 between indicated cell pairs in K10-rtTA; TRE-spastin epidermis. n = 40 pairs from 2 mice for each pair type. (D) Transmission electron micrographs of desmosomes in control and K10-rtTA; TRE-spastin epidermis. Scale-500nm. (E) A pair of spastin-positive cells in K14-rtTA; TRE-spastin epidermis showing that spastin expression does not intrinsically alter cortical desmoplakin localization. Scale-10µm. (F) Quantification of desmoplakin immunofluorescence at cell-cell boundaries between indicated cell pairs in K14-rtTA; TRE-spastin epidermis with sparse HA-spastin suprabasal cells. Control (n = 25 pairs), HA+/HA+ (n = 15 pairs), HA+/HA- (n = 26 pairs), and HA-/HA- (n = 36 pairs) from 2 mice for each pair type.
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 between indicated cell pairs in control or K14-rtTA; TRE-spastin epidermis with many suprabasal spastin-positive cells. Control (n = 33 pairs), HA+/HA+ (n = 32 pairs), HA+/HA- (n = 33 pairs), HA-/HA- (n = 28 pairs) from 2 mice for each cell pair. (C) Cortical DSG1 localization is maintained in individual spastin-positive suprabasal cells (indicated by asterisks). (D) Cortical DSG1 expression is perturbed in K14-rtTA; TRE-spastin epidermis when a large number of suprabasal cells overexpress spastin. All scale bars are 25 µm.
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 epidermis. Scale-500nm. (C) Examples of protein localization in the corneocytes of K10-rtTA; TRE-spastin mice. All images are inverted fluorescence (black indicates signal). All of the indicated proteins are absent in wild-type corneocytes. Scale-10µm. (D) Spastin expression cell-autonomously causes abnormal retention of cytoplasmic proteins. Scale-10μm. (E) Isolated corneocytes from control and K10-rtTA; TRE-spastin mice. Scale-25μm. (F) Quantification of isolated cornified envelopes. n = 40 random fields from 4 mice for each genotype. (G) X-gal barrier assay on e18.5 control and K10-rtTA; TRE-spastin embryos. (H) Epidermal cross-sections from e16.5 control and K10-rtTA TRE-spastin embryos, stained for the differentiation marker filaggrin. Scale-25μm. (I) Premature differentiation is non-cell autonomous in K10-rtTA; TRE-spastin epidermis. Filaggrin is induced in both spastin-positive and spastin-negative cells in prematurely differentiating K10-rtTA; TRE-spastin epidermis. Spastin-positive cells in the spinous layer do not induce filaggrin. Scale-25μm. (J) K10-rtTA; TRE-spastin e16.5 embryos prematurely form an epidermal barrier. Data are presented as mean ± S.E.M. ***p<0.001.
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 that ZO-1 is still cortically localized in spastin-positive cells. Scale-10µm. (C) Localization of occludin at the cell cortex is maintained in K10-rtTA; TRE-spastin epidermis. Scale-10µm. (D) Biotin diffusion is blocked by occludin in K10-rtTA; TRE-spastin epidermis. Scale-10µm.
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
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A transgenic toolkit for visualizing and perturbing microtubules reveals unexpected functions in the epidermis
eLife 6:e29834.
https://doi.org/10.7554/eLife.29834
