β-Spectrin Regulates the Hippo Signaling Pathway and Modulates the Basal Actin Network*

Background: The Hippo signaling pathway is regulated by mechanical signals from the cytoskeleton. Results: Perturbation of the spectrin cytoskeleton lead to loss of Hippo signaling phenotypes in Drosophila and mammalian cells. Conclusion: β-Spectrin regulates Hippo signaling activity in Drosophila and mammalian cells. Significance: The spectrin cytoskeleton can function as a tension-sensing system and feed into the Hippo pathway. Emerging evidence suggests functional regulation of the Hippo pathway by the actin cytoskeleton, although the detailed molecular mechanism remains incomplete. In a genetic screen, we identified a requirement for β-Spectrin in the posterior follicle cells for the oocyte repolarization process during Drosophila mid-oogenesis. β-spectrin mutations lead to loss of Hippo signaling activity in the follicle cells. A similar reduction of Hippo signaling activity was observed after β-Spectrin knockdown in mammalian cells. We further demonstrated that β-spectrin mutations disrupt the basal actin network in follicle cells. The abnormal stress fiber-like actin structure on the basal side of follicle cells provides a likely link between the β-spectrin mutations and the loss of the Hippo signaling activity phenotype.

The evolutionarily conserved Hippo signaling pathway has been discovered to have an essential function in organ size control (1). It was later found to also play important roles in processes such as differentiation and morphogenesis in various developmental contexts (1)(2)(3). The upstream regulatory network of the Hippo pathway is complex in contrast to its well defined core pathway components (2). Importantly, multiple lines of evidence have pointed to the actin cytoskeleton as an essential regulator of Hippo signaling activity (4 -10). These observations are consistent with the hypothesis that cells in tissues and organs can sense mechanical signals such as geometry and tension and that these mechanical signals are passed through the actin cytoskeleton to the Hippo pathway for coordination of cell behaviors. However, the mechanistic link between the actin cytoskeleton and the Hippo pathway remains largely unclear.
Spectrins, originally identified from human erythrocyte ghosts in 1968 (14), are filamentous proteins that are organized into a polygonal meshwork underneath the red blood cell membrane (15,16). The spectrin-based cytoskeleton confers high deformability and elasticity to the cell membrane for red blood cells to endure the mechanical and osmotic stresses as they circulate through the body. Defects in the spectrin network are associated with many forms of hemolytic anemia (17)(18)(19)(20). The spectrin cytoskeletal network exists in most metazoan cell types (21)(22)(23)(24), where it has been found to be important for the formation of cell membrane microdomains (25)(26)(27)(28), axonal growth (29,30), and synapse development (31)(32)(33)(34). Although spectrins exhibit diverse functions in different tissues and organs, the spectrin network seems to have a general structural and supporting function related to the mechanical properties of cells (24,35). A recent study has demonstrated that the spectrin cytoskeleton in the Caenorhabditis elegans touch sensory neurons is required for maintaining neuronal prestress status, protecting these neurons from mechanical stress, and enhancing the mechanical sensitivity of these neurons (36).
In this study, we demonstrate that ␤-spectrin mutations disrupt Hippo signaling activity in the follicular epithelial cells during Drosophila oogenesis. We also show that the requirement of ␤-Spectrin in the Hippo signaling pathway is conserved in mammalian cells. Although the link between actin stress fibers and YAP/TAZ activity has been established previously in mammalian cell cultures (4 -6, 9), here we are able to demonstrate a link between the formation of abnormal stress fibers and loss of Hippo signaling activity in ␤-spectrin mutant cells in an in vivo developmental context.
Mutation Mapping-The FY18 complementation group was mapped through recombination mapping to the region proximal of forked (f) at 15F4-F7. Duplication mapping showed that the FY18 complementation group could be rescued by Dp (1;Y)w73, which further narrowed the mutations to chromosomal region 15F-16F. The mutant alleles were then balanced over an FM7, KrϾGFP chromosome. The genomic DNA from homozygous mutant embryos was sequenced for mutations of candidate genes in the region. The sequence of the starting chromosome y w FRT19A was used as the reference sequence.
MCF10A cells were seeded in 6-well plates onto glass coverslips and cultured until the desired density was reached. Cells were then fixed and permeabilized with 4% formaldehyde and 0.1% Triton X-100, respectively. Next, MCF10A cells were blocked using 10% goat serum, followed by incubation with primary and secondary antibodies. The primary antibodies used were mouse YAP antibody (1:100, Santa Cruz Biotechnology) and rabbit polyclonal SPTBN2 antibody (1:100, Proteintech). All images were taken using a Zeiss fluorescence microscope and processed by Adobe software.
Time-lapse Imaging-The egg chambers were dissected and cultured for live imaging following the protocol described before (49). All time-lapse imaging was performed on a Leica TCS SP8 confocal microscope using a ϫ40 water immersion lens with a 488-nm optically pumped semiconductor lasers. Image z stacks were taken at 1-m steps from the egg chamber surface to 8 m below the surface. Images were taken at 30-s intervals from 30 min to 2 h. S2 Cell Culture, Luciferase Assay, and Quantitative RT-PCR-S2 cells were cultured at 28°C in Schneider's Drosophila medium (Invitrogen) supplemented with 10% fetal bovine serum and 0.5ϫ penicillin-streptomycin (Invitrogen). S2 cells were seeded in the presence of the indicated dsRNA, followed by transient transfection with the Yki-pMT, Renilla-pMT, and 3xSd2-Luciferase plasmids (gifts from Dr. Georg Halder) by Cellfectin II (Invitrogen). After 24 h, copper sulfate was added to a final concentration of 500 M, and luciferase activity was measured after 3-day incubation using the Promega Dual-Glo kit. The knockdown efficiency for ␤-spectrin was assayed by qRT-PCR. 2 The primer sequences for the ␤-spectrin gene were 5ЈACTTCGGTGCGTGCATCTCG3Ј and 5ЈTTGAT-GCGTTGTCGCTCAGC3Ј.

RESULTS
␤-spectrin Mutations in Posterior Follicle Cells (PFCs) Lead to Oocyte Polarity Defects during Mid-oogenesis-In a Drosophila egg chamber, the oocyte resides in a posterior position, surrounded by somatic epithelial follicle cells (FCs). The oocyte establishes the anterior-posterior axis and the dorsal-ventral axis polarity of the egg and embryo during oogenesis (50). The polarization processes are dependent on the interactions between the oocyte and the FCs. In particular, during midoogenesis, the oocyte dorsal-ventral asymmetry is established when an unknown signal from the PFCs triggers the oocyte cytoskeleton to repolarize, and then the oocyte nucleus migrates from the posterior end to the future dorsal-anterior corner of the oocyte (50). Multiple signaling pathway activities in the PFCs, including JAK/STAT (40), EGFR (51, 52), Notch (53,54), and Hippo (55)(56)(57), are required for this dorsal-ventral symmetry-breaking event during mid-oogenesis.
In a genetic screen for mutants affecting oocyte polarity (58), a complementation group of six lethal alleles was isolated as required in the PFCs for oocyte dorsal-ventral asymmetry formation. Through recombination and duplication mapping, we mapped the lethality of these alleles to chromosomal region 15F-16F. Sequencing of these alleles showed that they all contained mutations that lead to premature stop codons in the ␤-spectrin gene (Fig. 1A). All six alleles exhibited similar phenotypes with comparable severity and penetrance. We therefore used ␤-spectrin FY18 as a representative allele for the rest of the study. When the PFCs were homozygous mutant for ␤-spectrin, the oocyte nucleus frequently remained at the posterior end of the oocyte during mid-oogenesis ( Fig. 1, B and C, 35.8%, n ϭ 229). To confirm that ␤-spectrin mutations lead to a general disruption of oocyte polarity instead of specifically affecting the oocyte nucleus migration event, we further examined a panel of oocyte polarity markers. We first examined a microtubule polarity marker, Kinesin-␤-gal. Kinesin-␤-gal forms a crescent at the posterior end of the oocyte in a wild-type egg chamber after mid-oogenesis (38) (Fig. 1B). Kinesin-␤-gal appeared frequently in aggregates or as a diffused cloud in the center of the oocyte in the presence of ␤-spectrin mutant PFCs (Fig. 1C, 85.4%, n ϭ 64). A second oocyte polarity marker we examined was Staufen. Staufen typically localizes at the posterior end of the oocyte after mid-oogenesis for anchoring the maternal RNAs (59) (Fig. 1D). When the PFCs were mutant for ␤-spectrin, Staufen also appeared dispersed in the center of the oocyte (Fig. 1E, 91.1%, n ϭ 90). Taken together, these results indicate that ␤-Spectrin is required in the PFCs for the oocyte polarization process during mid-oogenesis.
The Drosophila genome contains three spectrin genes, including ␣-spectrin (60), ␤-spectrin (61) and ␤-heavy-spectrin  (75). CH, Calponin homology domain; PH, pleckstrin homology domain. B and C, a wild-type egg chamber (B) and an egg chamber with ␤-spectrin FY18 mutant PFCs (C) expressing Kinesin-␤-gal are stained for ␤-gal (red) and DNA (blue). In the stage 8 wild-type egg chamber, the oocyte nucleus migrates to the dorsal-anterior corner (B, blue; B", gray, white arrowhead), and Kinesin-␤-gal forms a crescent at the posterior of the oocyte (B, red; B', gray, red arrowhead). In the egg chamber containing ␤-spectrin FY18 mutant PFCs, the oocyte nucleus stays at the posterior (C, blue; C", gray, white arrowhead), and Kinesin-␤-gal (Kin-␤-gal) congregates at the center of the oocyte (C, red; C', gray, red arrowhead). D and E, a wild-type egg chamber (D) and an egg chamber with ␤-spectrin FY18 mutant PFCs (E) expressing Staufen-GFP (Stau-GFP, green) and stained for DNA (blue). In the wild-type egg chamber, Staufen-GFP is tightly localized at the posterior of the oocyte (red arrowhead). In the egg chamber containing ␤-spectrin FY18 mutant PFCs, Staufen-GFP appears dispersed in the center of the oocyte (red arrowhead). F, an egg chamber with follicle cells expressing an RNAi construct against ␣-spectrin stained for Gurken (Grk, red) and DNA (green). Note that the oocyte nucleus (white arrowhead) stays at the posterior end of the oocyte. Grk is mislocalized at the posterior end of the oocyte together with the oocyte nucleus. G, an egg chamber with ␤-spectrin FY18 mutant PFCs stained for Fas II (G, red; G', gray) and DNA (blue). The posterior polar cells marked by Fas II staining (red arrowhead) remain in direct contact with the oocyte, whereas the oocyte polarity is abnormal, marked by the posterior localization of the oocyte nucleus. H, an egg chamber with follicle cells expressing an RNAi construct against ␣-spectrin stained for Fas II (red) and DNA (green). Note that the oocyte remains in direct contact with the posterior polar cells marked by Fas II (red arrowhead). (62). It has been shown previously that ␣-spectrin mutations in the PFCs lead to disruption of oocyte polarity (63). In the report, the authors attributed the oocyte polarity defects to a loss of direct contact between the polar follicle cells and the oocyte, caused by hyperplasia of the ␣-spectrin mutant cells. Although in our experiments 55.9% of the ␤-spectrin mutant PFCs exhibited a hyperplasia phenotype (n ϭ 228), a significant portion of the ␤-spectrin mutant cells maintained a monolayered or bilayered epithelial structure even though they exhibited an oocyte polarity defect. When we marked the posterior polar cells with Fas II, in 25 of 27 egg chambers where we observed oocyte polarity defects in the presence of ␤-spectrin mutant PFCs, the posterior polar cells remained in close contact with the oocyte (Fig. 1G). Similarly, when we knocked down ␣-spectrin in the follicle cells by RNAi, we observed oocyte polarity defects similar to those in the ␤-spectrin mutants (Fig. 1F). Nevertheless, in these cases, the oocyte was frequently observed as being in direct contact with the posterior polar cells (Fig. 1H). Therefore, we concluded that the oocyte polarity defects in ␤-spectrin mutants are unlikely to be caused by the separation of the posterior polar cells and the oocyte.

␤-Spectrin Regulates the Hippo Signaling Pathway
␤-spectrin Mutations Lead to Loss of Hippo Signaling Activity in Follicle Cells-The oocyte polarity defects in the presence of ␤-spectrin mutant PFCs suggested that ␤-spectrin mutations might affect one (or more) of the four signaling pathways required in the PFCs for the oocyte polarization process, namely the JAK/STAT, EGFR, Notch, and Hippo signaling pathways. We therefore examined well established signaling reporters for each of these pathways.
Next, we examined the expression of a kekkon-lacZ (kek-lacZ) reporter, which is normally induced in PFCs in response to EGFR signaling activation during mid-oogenesis (41) (Fig.  2C). The kek-lacZ reporter was activated when the PFCs were mutant for ␤-spectrin (Fig. 2D), indicating that EGFR signaling was also unaffected by ␤-spectrin mutations.
Notch signaling activity is required for the follicle cells to undergo a mitotic cycle-to-endocycle switch during mid-oogenesis (53,54). When Notch signaling activity is compromised in follicle cells, the follicle cells continue to divide and fail to differentiate properly along the oocyte anterior-posterior axis.
In the presence of loss of function Notch signaling mutant PFCs, oocyte polarity is also disrupted. We therefore examined the expression of Cut, a Notch signaling activity target. Cut expression is normally repressed by Notch signaling activity after stage 6 of oogenesis (Fig. 2E) (64). The ␤-spectrin mutant PFCs frequently retained a high level of Cut expression (Fig. 2F,  59.3%, n ϭ 27). It is noteworthy that ␤-spectrin mutant FCs on the lateral side of the egg chamber did not retain a high Cut expression level (Fig. 2F). In other words, ␤-spectrin mutations affected Notch signaling specifically in PFCs, unlike the core Notch signaling pathway mutants, which affect all follicle cells regardless of position along the oocyte anterior-posterior axis. It has been shown previously that such a PFC-specific loss of Notch signaling phenotype is typically exhibited in Hippo pathway mutants (55)(56)(57). Therefore, we examined the Hippo signaling activity reporters expanded-lacZ (ex-lacZ) (65) and diap1-lacZ (66) in follicle cells. Hippo signaling activity normally represses the expression of expanded-lacZ (Fig. 2G). The ␤-spectrin mutant clones frequently showed elevated expression levels of ex-lacZ, regardless of the clone position in the egg chamber (Fig. 2H, 67.6%, n ϭ 37). Similarly, the expression level of diap1-lacZ was also up-regulated in ␤-spectrin mutant cells (Fig. 2I, 69.2%, n ϭ 26). Moreover, Hippo signaling activity has been shown recently to be required for border cell migration in the follicular epithelium (67). When border cells were mutant for ␤-spectrin (Fig. 2J) or when ␣-spectrin was knocked down in border cells by RNAi (Fig. 2K), the border cells exhibited migration defects characteristic of those produced by the Hippo pathway mutants. Taken together, our results indicated that ␤-spectrin mutations lead to oocyte polarity defects through disrupting Hippo signaling activity in follicle cells.
To test whether the function of ␤-Spectrin in the Hippo signaling pathway is specific to the follicular epithelium in Drosophila, we first examined spectrin function in the imaginal discs from third-instar larvae. We checked the expression of two Hippo signaling reporters, cyclin E-lacZ and diap1-lacZ (66), in the eye and wing imaginal discs. We found that the expression level of these reporters in the ␤-spectrin mutant cells was comparable with that of wild-type cells (data not shown), indicating that ␤-spectrin mutations do not significantly affect Hippo signaling activity in the eye and wing imaginal discs. Interestingly, when we knocked down ␣-spectrin in the wing discs through RNAi, we observed a 1.23-fold increase in adult wing size in comparison with the control (Fig. 3, A-E), indicating an effect on growth control, most likely via an effect on the Hippo signaling pathway. ␤-Spectrin is specifically localized to

␤-Spectrin Regulates the Hippo Signaling Pathway
the lateral domain of epithelial cells, whereas ␣-Spectrin is localized both apically and laterally (63,68). Therefore, depletion of ␣-Spectrin by RNAi might have a stronger effect on the disruption of the spectrin network and Hippo signaling activity in the imaginal disc cells.
In Drosophila S2 cells where Yorkie (Yki) was expressed under the control of a methallothionein promoter, the luciferase activity of a reporter, 3XSd2-Luc, was used successfully to monitor Yki activity (8). When we treated these cells with dsRNA against ␤-spectrin, we observed a 1.4-fold increase in relative luciferase activity compared with that of the DMSOtreated control group. The increase was comparable with that of the positive control expanded (ex) and capping protein ␣ (cpa) RNAi (Fig. 3, F and G). These results indicated that the requirement of ␤-Spectrin for Hippo signaling activity is not limited to the follicle cells in Drosophila.
␤-Spectrin Is Required for Hippo Signaling Activity in Mammalian Cells-Two recent affinity purification and MS-based proteomic studies have included spectrins in the Hippo pathway protein-protein interaction network (69,70). Specifically, in cultured human cells, an interaction between the bait protein Salvador homolog 1 (SAV1) and SPTAN1, a human ␣-Spectrin isoform, has been reported (69). Therefore, we wanted to test whether ␤-Spectrin has a conserved role in regulating the Hippo pathway in mammals.
We tested the effect of ␤-spectrin knockdown on Hippo signaling activity in human mammary epithelial MCF10A cells. When MCF10A cells are grown at a low cell density, the YAP/ TAZ proteins (the mammalian homologs of Yorkie) localize in the cell nucleus and promote proliferation. When MCF10A cell culture reaches a high cell density, the YAP/TAZ proteins translocate to the cytoplasm, and cell proliferation is inhibited (9). In human cells, five genes, SPTB, SPTBN1, SPTBN2, SPTBN4, and SPTBN5, encode ␤-spectrins. On the basis of previous studies (71), we focused on SPTBN1, SPTBN2, and SPTBN4, which are most likely to be functional in MCF10A epithelial cells because SPTB and SPTBN5 are known to be more restrictively expressed in neuronal cells and other specific cell types (71). Microarray gene expression profiling of MCF10A cells further demonstrated that SPTBN2 is most highly expressed among the three ␤-spectrin genes (Fig. 4A). Therefore, we chose to knock down SPTBN2 to investigate the potential role of ␤-spectrin in regulating Hippo signaling activity. MCF10A cells were transduced with lentiviruses containing a scrambled control shRNA construct or three different SPTBN2-targeting shRNA constructs, and the knockdown efficiency was confirmed by qRT-PCR (Fig. 4B) and Western blotting (Fig. 4C). In control cells, SPTBN2 is mostly localized on the cytoplasmic membrane, although the antibody against SPTBN2 also shows nonspecific nucleus staining (Fig. 4D). The membrane localization of SPTBN2 was lost in most cells from the SPTBN2 knockdown population (denoted as SPTBN2 lo cells) (Fig. 4D'). However, the knockdown was less efficient in a few cells so that they maintained SPTBN2 membrane staining (denoted as SPTBN2 hi cells) (Fig. 4D', arrowhead). To normalize the effect of local cell density on YAP localization as mentioned previously, we randomly picked clusters of SPTBN2expressing SPTBN2 hi cells in the knockdown population and compared them with their neighboring SPTBN2 lo cells. As shown in Fig. 4, E-H, compared with SPTBN2 lo cells, SPTBN2 hi cells showed reduced nuclear localization of YAP, indicating that SPTBN2 regulates YAP localization and activity in MCF10A cells.
␤-spectrin Mutations Lead to Assembly of Abnormal Actin Stress Fibers in the Follicle Cells-Hippo signaling activity is regulated by a plethora of upstream components, including cell polarity components, cell-cell adhesion molecules, G proteincoupled receptor signaling, and the actin cytoskeleton (2). To understand how ␤-Spectrin affects Hippo signaling activity, we examined major upstream regulators in ␤-spectrin mutant cells.
␤-Spectrin is specifically localized to the lateral domain of the follicle cells (63) (Fig. 5, A and B). It is possible that ␤-Spectrin might affect the Hippo signaling pathway through disruption of the cell polarity system. We first examined localization of the cell apical components such as ␣-Spectrin and aPKC. Both ␣-Spectrin and aPKC remained apically localized in ␤-spectrin mutant follicle cells (Fig. 5, C and D). We also examined the apically localized protein Merlin and Expanded, two known upstream regulators of the Hippo signaling pathway. Similarly, the apical localization of Merlin and Expanded remained unchanged in the ␤-spectrin mutant cells (Fig. 5, E-G). Next, we examined the adhesion junctional components Ecadherin (Ecad) and Armadillo (Arm, the Drosophila ␤-Catenin homolog). Similarly, the junctional localization of Ecad and Arm was largely unaffected in ␤-spectrin mutant cells (Fig. 5, H and I). Third, we examined basolateral proteins such as Discs large (Dlg), Na ϩ , and K ϩ -ATPase, and their localization was also unaffected in the ␤-spectrin mutant cells (Fig. 5, J  and K). These results suggest that, although ␤-Spectrin is part of the cell polarity system, mutations in ␤-spectrin do not disrupt general apical-basal polarity in follicle cells.
Spectrins are well known actin cross-linking proteins (11)(12)(13). It is possible that ␤-spectrin mutations might affect the actin cytoskeletal structure in follicle cells. Interestingly, phalloidin staining revealed an abnormal actin cytoskeletal struc-  MARCH 6, 2015 • VOLUME 290 • NUMBER 10 ture on the basal side of ␤-spectrin mutant follicle cells (Fig. 5,  L-O). The actin filaments on the basal side of wild-type follicle cells are oriented perpendicularly to the anterior-posterior axis of the egg chamber after mid-oogenesis (72). These bundled actin filaments, which are part of the planar cell polarity system in the follicular epithelium, resemble stress fibers in cultured cells. In the ␤-spectrin mutant cells, the actin filaments lose the planar polarized orientation (Fig. 5, L and M) and frequently form actin patches with intensified phalloidin staining on the basal side of the epithelium (Fig. 5, N and O). We further analyzed the actin dynamics in the ␤-spectrin mutant cells through time-lapse imaging of cultured live stage 8 -9 egg chambers. The F-actin filaments were visualized using an Utrophin-GFP marker (45). Although the basal F-actin filaments appear relatively stable in wild-type follicle cells, as reported previously (supplemental Movie 1) (73), in ␤-spectrin mutant cells, the F-actin filaments failed to assemble into parallel bundles and, instead, initiated radial contractions over time (supplemental Movie 2).

DISCUSSION
The Link between the Basal F-actin Phenotype and the Hippo Signaling Defects in ␤-spectrin Mutant Cells-Multiple lines of evidence in cultured cells have linked actin stress fibers and YAP/TAZ activity. For example, YAP/TAZ nuclear localization coincided with the presence of abundant stress fibers in cultured cells (5). YAP/TAZ activity decreased in cells treated with the actin depolymerization drugs cytochalasin D and latrunculin A/B, which also reduced stress fiber formation (5,6). YAP/ TAZ activity increased in cultured cells overexpressing Diaphanous, which induced stress fiber formation (4,8). Several actin binding proteins, including Cofilin 1/2, Capzb, and Gelsolin, were identified as inhibitors of YAP/TAZ activity. Depletion of these proteins caused an increase in both stress fiber formation and YAP/TAZ activity (9).
Follicle cells maintain basal F-actin filaments structurally similar to stress fibers in cultured cells (72). In live ␤-spectrin mutant cells, the F-actin filaments fail to assemble into a planar polarized network and seem to undergo radial contractions, possibly as a result of repeated actin assembly-disassembly cycles. In the fixed ␤-spectrin mutant cells, we observed basal actin patches characterized by intense phalloidin staining and lack of planar polarized organization. In light of previous observations in cell cultures linking the formation of actin stress fibers and YAP/TAZ activity, it seems likely that the basal actin phenotype and the Hippo signaling phenotype in the ␤-spectrin mutant cells are tightly linked.
Spectrins as General Tension-sensing Molecules underneath the Cell Membrane-The Hippo signaling pathway is known to link with the cytoskeletal tension in cultured cells (2). Importantly, it has been shown recently that actomyosin-based cellular tension is critical for Hippo signaling activity in vivo (10).
Spectrins are long and flexible molecules that form a network underneath the cell membrane. The spectrin function is well characterized in red blood cells as providing cells with mechanical properties, including elasticity and deformability, that allow these cells to survive circulation (74). In other cell types, the spectrin function is much less clear. It has been shown that cytoskeletal tension regulates Hippo activity via promoting Ajuba and Wts complex formation at the adherent junctions in a tension-dependent manner (10). We examined Ajuba and Wts complex localization in follicle cells, and they are both junctionally localized in the ␤-spectrin mutant cells. However, using confocal microscopy, we did not reliably detect a difference of Wts/Ajuba junctional fluorescence intensity between ␤-spectrin mutant cells and wild-type follicle cells (data not shown). It is possible that the spectrin cytoskeleton is part of a cellular tension-sensing system that feeds into the Hippo regulation network. Although we demonstrated a link between spectrin function and actin network organization, a possible link between spectrin function and myosin-based cellular tension still needs further investigation.