Espin cross-links cause the elongation of microvillus-type parallel actin bundles in vivo - PubMed (original) (raw)
Espin cross-links cause the elongation of microvillus-type parallel actin bundles in vivo
Patricia A Loomis et al. J Cell Biol. 2003.
Abstract
The espin actin-bundling proteins, which are the target of the jerker deafness mutation, caused a dramatic, concentration-dependent lengthening of LLC-PK1-CL4 cell microvilli and their parallel actin bundles. Espin level was also positively correlated with stereocilium length in hair cells. Villin, but not fascin or fimbrin, also produced noticeable lengthening. The espin COOH-terminal peptide, which contains the actin-bundling module, was necessary and sufficient for lengthening. Lengthening was blocked by 100 nM cytochalasin D. Espin cross-links slowed actin depolymerization in vitro less than twofold. Elimination of an actin monomer-binding WASP homology 2 domain and a profilin-binding proline-rich domain from espin did not decrease lengthening, but made it possible to demonstrate that actin incorporation was restricted to the microvillar tip and that bundles continued to undergo actin treadmilling at approximately 1.5 s-1 during and after lengthening. Thus, through relatively subtle effects on actin polymerization/depolymerization reactions in a treadmilling parallel actin bundle, espin cross-links cause pronounced barbed-end elongation and, thereby, make a longer bundle without joining shorter modules.
Figures
Figure 1.
Espin increases the length of microvilli in transfected CL4 cells. CL4 cells were transfected with GFP-espin cDNA, double labeled for F-actin with Texas red–phalloidin and examined by confocal microscopy. Images of the apical surface of the monolayer are shown from (A) above or (B) the side (different fields). The GFP-espin and Texas red–phalloidin are colocalized (yellow) in microvillus-like projections that are much longer than the brush border microvilli (red) of surrounding control cells. Bars, 5 μm.
Figure 2.
Microvillar PAB length is positively correlated with espin level. CL4 cells were transfected with GFP-espin cDNA using a series of CMV promoter deletion constructs. Microvillar PAB length was measured by confocal microscopy (n = 187–555 microvilli, 12–28 cells), and replicate dishes were dissolved in SDS gel sample buffer to measure relative espin level by Western blotting (100% = full strength CMV promoter). (A) Frequency plots of microvillar PAB length for control cells and for cells transfected with two of the espin constructs. (B) Espin concentration dependence of microvillar PAB lengthening (inset, Western blot from duplicate dishes). Error bars, SEM. All groups were significant compared with control: one-way ANOVA (F(4,1615) = 1576, P < 0.0001); Dunnett's test (P < 0.01).
Figure 3.
The long microvilli elicited by espin contain collections of continuous actin filaments of the correct polarity. CL4 cells were transfected with GFP-espin cDNA, fixed, and processed for EM (A and B) without or (C–E) with treatment with Triton X-100 and labeling with S1. Bars: (A, C, and E) 100 nm; (B and D) 50 nm.
Figure 4.
Espin level is positively correlated with stereocilium length in cochlear hair cells. (Top) Diagram of cochlear whole mount showing the three rows of outer hair cells (OHC) and one row of inner hair cells (IHC) separated by pillar cells (PC). Confocal images of hair cells in the (A and B) apical, (D) middle, and (F) basal turns of a single cochlear whole mount labeled by immunofluo-rescence using espin antibody were collected using identical settings. Specimen orientation dictated that the image of the inner hair cells in the (A) apical turn be collected separately from that of the (B) outer hair cells. Increases in fluorescence intensity from basal turn to apical turn are also evident in the corresponding three-dimensional color-scale intensity plots shown at the right (C, E, and G: red, high espin; blue, low espin). Bar, 5 μm.
Figure 5.
The espin COOH-terminal actin-bundling module is required for PAB lengthening. CL4 cells were transfected with the GFP-espin truncation or deletion constructs shown in the stick figure diagrams. Microvillar PAB length was measured by confocal microscopy (mean ± SEM; n = 143–272 microvilli, 11–17 cells), and GFP-espin protein levels in replicate dishes were compared by Western blotting using GFP antibody (inset, sample Western blots for three constructs shown in duplicate). PR1, proline-rich peptide 1; xAB, additional F-actin–binding site; PR2, proline-rich peptide 2; WH2, WASP homology 2; ABM, actin-bundling module. ΔC117, the espin construct missing the actin-bundling module, is panel i.
Figure 6.
Significant microvillar PAB lengthening activity is observed for espin isoforms, the espin COOH-terminal peptide, villin and T-fimbrin, but not fascin. CL4 cells were transfected with the designated actin-bundling protein construct (SC espin, Sertoli cell espin). (A) Stick figure diagram of espin constructs. See Fig. 5 legend for domain abbreviations. Asterisks indicate peptides encoded by exons unique to small espin that bracket the WH2 domain. (B and C) Microvillar PAB length was measured by confocal microscopy, and protein levels were compared by Western blotting replicate dishes using espin COOH-terminal peptide antibody (B, top) or GFP antibody (B, bottom left and right). Length measurements were corrected for differences in level of protein accumulation using the espin concentration dependence (Fig. 2 B) and are plotted in C as a percentage of the microvillar PAB lengthening activity expected for espin when expressed at the same level (mean ± SEM; n = 135–254 microvilli, 10–15 cells). All groups were significant compared with control, except S39A-fascin: one-way ANOVA (F(6,1533) = 1166, P < 0.0001); Dunnett's test (P < 0.01). (D–G) Confocal images of the apical surface of CL4 cell monolayer showing fluorescent phalloidin localization in cells expressing (D) espin, (E) small espin, (F) S39A fascin, and (G) villin. Bar, 5 μm.
Figure 7.
Espin causes a <**2-fold decrease in actin depolymerization in vitro and binds actin monomer and profilin in pull-down assays.** (A) Release of actin monomer into the supernatant after the addition of 10 μM latrunculin A to preformed actin filaments that had been incubated in the absence (Control) or presence of espin to provide a maximum level of cross-links (+Espin). (B) Release of 3H-nucleotide into the supernatant after the addition of an unlabeled ATP chase to actin filaments that had been polymerized in the presence of [3H]ATP and incubated in the absence (Control) or presence of espin to provide a maximum level of cross-links (+Espin). In some experiments, 3 μM human cofilin was added 20 min before the unlabeled ATP (mean ± SEM; _n_ = 3). (C) Binding of G-actin to GST-espin (Espin) or GST-espin missing the WH2 consensus domain (ΔWH2) in a pull-down assay (o, GST-espin construct; >, G-actin). ∼20% of the G-actin added bound to GST-espin. (D) Binding of 6X His-tagged espin constructs to GST-profilin IIa, GST-profilin I, or GST in a pull-down assay. Espin is compared with deletion constructs missing the NH2-terminal (ΔPR1) or COOH-terminal (ΔPR2) proline-rich peptide or both proline-rich peptides (ΔPR1+2).
Figure 8.
FRAP and rhodamine-actin decoration reveal actin incorporation at the microvillar tip and actin treadmilling in the long microvilli of espin-expressing CL4 cells. (A–C) Examples of GFP–β-actin FRAP in microvilli of CL4 cells expressing espin (A), the ΔP8ΔWH2 espin construct (B), or no espin (C). Recovery times are shown. Arrowhead in C represents a microvillus as it enters field and obscures recovery. Bar, 1 μm. (D–G) Rhodamine-actin labeling in the microvilli of detergent-permeabilized CL4 cells (D and E) 4 h or (F and G) 24 h after transfection with the ΔP8ΔWH2 espin construct (E and G, with fluorescein-phalloidin counterstain). Bar, 2 μm.
References
- Bartles, J.R., A. Wierda, and L. Zheng. 1996. Identification and characterization of espin, an actin-binding protein localized to the F-actin-rich junctional plaques of Sertoli cell ectoplasmic specializations. J. Cell Sci. 109:1229–1239. - PubMed
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