Crumbs and the apical spectrin cytoskeleton regulate R8 cell fate in the Drosophila eye.

The Hippo pathway is an important regulator of organ growth and cell fate. In the R8 photoreceptor cells of the Drosophila melanogaster eye, the Hippo pathway controls the fate choice between one of two subtypes that express either the blue light-sensitive Rhodopsin 5 (Hippo inactive R8 subtype) or the green light-sensitive Rhodopsin 6 (Hippo active R8 subtype). The degree to which the mechanism of Hippo signal transduction and the proteins that mediate it are conserved in organ growth and R8 cell fate choice is currently unclear. Here, we identify Crumbs and the apical spectrin cytoskeleton as regulators of R8 cell fate. By contrast, other proteins that influence Hippo-dependent organ growth, such as the basolateral spectrin cytoskeleton and Ajuba, are dispensable for the R8 cell fate choice. Surprisingly, Crumbs promotes the Rhodopsin 5 cell fate, which is driven by Yorkie, rather than the Rhodopsin 6 cell fate, which is driven by Warts and the Hippo pathway, which contrasts with its impact on Hippo activity in organ growth. Furthermore, neither the apical spectrin cytoskeleton nor Crumbs appear to regulate the Hippo pathway through mechanisms that have been observed in growing organs. Together, these results show that only a subset of Hippo pathway proteins regulate the R8 binary cell fate decision and that aspects of Hippo signalling differ between growing organs and post-mitotic R8 cells.

A Fresh Look at the Structure, Regulation, and Functions of Fodrin.

Fodrin and its erythroid cell-specific isoform spectrin are actin-associated fibrous proteins that play crucial roles in the maintenance of structural integrity in mammalian cells, which is necessary for proper cell function. Normal cell morphology is altered in diseases such as various cancers and certain neuronal disorders. Fodrin and spectrin are two-chain (αß) molecules that are encoded by paralogous genes and share many features but also demonstrate certain differences. Fodrin (in humans, typically a heterodimer of the products of the SPTAN1 and SPTBN1 genes) is expressed in nearly all cell types and is especially abundant in neuronal tissues, whereas spectrin (in humans, a heterodimer of the products of the SPTA1 and SPTB1 genes) is expressed almost exclusively in erythrocytes. To fulfill a role in such a variety of different cell types, it was anticipated that fodrin would need to be a more versatile scaffold than spectrin. Indeed, as summarized here, domains unique to fodrin and its regulation by Ca2+, calmodulin, and a variety of posttranslational modifications (PTMs) endow fodrin with additional specific functions. However, how fodrin structural variations and misregulated PTMs may contribute to the etiology of various cancers and neurodegenerative diseases needs to be further investigated.

The axonal actin-spectrin lattice acts as a tension buffering shock absorber.

Axons span extreme distances and are subject to significant stretch deformations during limb movements or sudden head movements, especially during impacts. Yet, axon biomechanics, and its relation to the ultrastructure that allows axons to withstand mechanical stress, is poorly understood. Using a custom developed force apparatus, we demonstrate that chick dorsal root ganglion axons exhibit a tension buffering or strain-softening response, where its steady state elastic modulus decreases with increasing strain. We then explore the contributions from the various cytoskeletal components of the axon to show that the recently discovered membrane-associated actin-spectrin scaffold plays a prominent mechanical role. Finally, using a theoretical model, we argue that the actin-spectrin skeleton acts as an axonal tension buffer by reversibly unfolding repeat domains of the spectrin tetramers to release excess mechanical stress. Our results revise the current viewpoint that microtubules and their associated proteins are the only significant load-bearing elements in axons.

Defining new mechanistic roles for αII spectrin in cardiac function.

Spectrins are cytoskeletal proteins essential for membrane biogenesis and regulation and serve critical roles in protein targeting and cellular signaling. αII spectrin (SPTAN1) is one of two α spectrin genes and αII spectrin dysfunction is linked to alterations in axon initial segment formation, cortical lamination, and neuronal excitability. Furthermore, human αII spectrin loss-of-function variants cause neurological disease. As global αII spectrin knockout mice are embryonic lethal, the in vivo roles of αII spectrin in adult heart are unknown and untested. Here, based on pronounced alterations in αII spectrin regulation in human heart failure we tested the in vivo roles of αII spectrin in the vertebrate heart. We created a mouse model of cardiomyocyte-selective αII spectrin-deficiency (cKO) and used this model to define the roles of αII spectrin in cardiac function. αII spectrin cKO mice displayed significant structural, cellular, and electrical phenotypes that resulted in accelerated structural remodeling, fibrosis, arrhythmia, and mortality in response to stress. At the molecular level, we demonstrate that αII spectrin plays a nodal role for global cardiac spectrin regulation, as αII spectrin cKO hearts exhibited remodeling of αI spectrin and altered ß-spectrin expression and localization. At the cellular level, αII spectrin deficiency resulted in altered expression, targeting, and regulation of cardiac ion channels NaV1.5 and KV4.3. In summary, our findings define critical and unexpected roles for the multifunctional αII spectrin protein in the heart. Furthermore, our work provides a new in vivo animal model to study the roles of αII spectrin in the cardiomyocyte.

Critical role of spectrin in hearing development and deafness.

Inner ear hair cells (HCs) detect sound through the deflection of mechanosensory stereocilia. Stereocilia are inserted into the cuticular plate of HCs by parallel actin rootlets, where they convert sound-induced mechanical vibrations into electrical signals. The molecules that support these rootlets and enable them to withstand constant mechanical stresses underpin our ability to hear. However, the structures of these molecules have remained unknown. We hypothesized that αII- and ßII-spectrin subunits fulfill this role, and investigated their structural organization in rodent HCs. Using super-resolution fluorescence imaging, we found that spectrin formed ring-like structures around the base of stereocilia rootlets. These spectrin rings were associated with the hearing ability of mice. Further, HC-specific, ßII-spectrin knockout mice displayed profound deafness. Overall, our work has identified and characterized structures of spectrin that play a crucial role in mammalian hearing development.

Whole-exome sequencing enables correct diagnosis and surgical management of rare inherited childhood anemia.

Correct diagnosis of inherited bone marrow failure syndromes is a challenge because of the significant overlap in clinical presentation of these disorders. Establishing right genetic diagnosis is crucial for patients' optimal clinical management and family counseling. A nondysmorphic infant reported here developed severe transfusion-dependent anemia and met clinical criteria for diagnosis of Diamond-Blackfan anemia (DBA). However, whole-exome sequencing demonstrated that the child was a compound heterozygote for a paternally inherited pathogenic truncating variant (SPTA1c.4975 C>T) and a novel maternally inherited missense variant of uncertain significance (SPTA1c.5029 G>A) within the spectrin gene, consistent with hereditary hemolytic anemia due to disruption of red blood cell (RBC) cytoskeleton. Ektacytometry demonstrated abnormal membrane flexibility of the child's RBCs. Scanning electron microscopy revealed morphological aberrations of the patient's RBCs. Both parents were found to have mild hereditary elliptocytosis. Importantly, patients with severe RBC membrane defects may be successfully managed with splenectomy to minimize peripheral destruction of misshapen RBCs, whereas patients with DBA require lifelong transfusions, steroid therapy, or hematopoietic stem cell transplantation. As suggested by the WES findings, splenectomy rendered our patient transfusion-independent, improving the family's quality of life and preventing transfusion-related iron overload. This case illustrates the utility of whole-exome sequencing in clinical care of children with genetic disorders of unclear presentation.

The tactile receptive fields of freely moving Caenorhabditis elegans nematodes.

Sensory neurons embedded in skin are responsible for the sense of touch. In humans and other mammals, touch sensation depends on thousands of diverse somatosensory neurons. By contrast, Caenorhabditis elegans nematodes have six gentle touch receptor neurons linked to simple behaviors. The classical touch assay uses an eyebrow hair to stimulate freely moving C. elegans, evoking evasive behavioral responses. This assay has led to the discovery of genes required for touch sensation, but does not provide control over stimulus strength or position. Here, we present an integrated system for performing automated, quantitative touch assays that circumvents these limitations and incorporates automated measurements of behavioral responses. The Highly Automated Worm Kicker (HAWK) unites a microfabricated silicon force sensor holding a glass bead forming the contact surface and video analysis with real-time force and position control. Using this system, we stimulated animals along the anterior-posterior axis and compared responses in wild-type and spc-1(dn) transgenic animals, which have a touch defect due to expression of a dominant-negative α-spectrin protein fragment. As expected from prior studies, delivering large stimuli anterior and posterior to the mid-point of the body evoked a reversal and a speed-up, respectively. The probability of evoking a response of either kind depended on stimulus strength and location; once initiated, the magnitude and quality of both reversal and speed-up behavioral responses were uncorrelated with stimulus location, strength, or the absence or presence of the spc-1(dn) transgene. Wild-type animals failed to respond when the stimulus was applied near the mid-point. These results show that stimulus strength and location govern the activation of a characteristic motor program and that the C. elegans body surface consists of two receptive fields separated by a gap.

Critical roles of αII spectrin in brain development and epileptic encephalopathy.

The nonerythrocytic α-spectrin-1 (SPTAN1) gene encodes the cytoskeletal protein αII spectrin. Mutations in SPTAN1 cause early infantile epileptic encephalopathy type 5 (EIEE5); however, the role of αII spectrin in neurodevelopment and EIEE5 pathogenesis is unknown. Prior work suggests that αII spectrin is absent in the axon initial segment (AIS) and contributes to a diffusion barrier in the distal axon. Here, we have shown that αII spectrin is expressed ubiquitously in rodent and human somatodendritic and axonal domains. CRISPR-mediated deletion of Sptan1 in embryonic rat forebrain by in utero electroporation caused altered dendritic and axonal development, loss of the AIS, and decreased inhibitory innervation. Overexpression of human EIEE5 mutant SPTAN1 in embryonic rat forebrain and mouse hippocampal neurons led to similar developmental defects that were also observed in EIEE5 patient-derived neurons. Additionally, patient-derived neurons displayed aggregation of spectrin complexes. Taken together, these findings implicate αII spectrin in critical aspects of dendritic and axonal development and synaptogenesis, and support a dominant-negative mechanism of SPTAN1 mutations in EIEE5.

The role of ßII spectrin in cardiac health and disease.

Spectrins are large, flexible proteins comprised of α-ß dimers that are connected head-to-head to form the canonical heterotetrameric spectrin structure. Spectrins were initially believed to be exclusively found in human erythrocytic membrane and are highly conserved among different species. ßII spectrin, the most common isoform of non-erythrocytic spectrin, is found in all nucleated cells and forms larger macromolecular complexes with ankyrins and actins. Not only is ßII spectrin a central cytoskeletal scaffolding protein involved in preserving cell structure but it has also emerged as a critical protein required for distinct physiologic functions such as posttranslational localization of crucial membrane proteins and signal transduction. In the heart, ßII spectrin plays a vital role in maintaining normal cardiac membrane excitability and proper cardiac development during embryogenesis. Mutations in ßII spectrin genes have been strongly linked with the development of serious cardiac disorders such as congenital arrhythmias, heart failure, and possibly sudden cardiac death. This review focuses on our current knowledge of the role ßII spectrin plays in the cardiovascular system in health and disease and the potential future clinical implications.

αII-spectrin in T cells is involved in the regulation of cell-cell contact leading to immunological synapse formation?

T-lymphocyte activation after antigen presentation to the T-Cell Receptor (TCR) is a critical step in the development of proper immune responses to infection and inflammation. This dynamic process involves reorganization of the actin cytoskeleton and signaling molecules at the cell membrane, leading to the formation of the Immunological Synapse (IS). The mechanisms regulating the formation of the IS are not completely understood. Nonerythroid spectrin is a membrane skeletal protein involved in the regulation of many cellular processes, including cell adhesion, signaling and actin cytoskeleton remodeling. However, the role of spectrin in IS formation has not been explored. We used molecular, imaging and cellular approaches to show that nonerythroid αII-spectrin redistributes to the IS during T-cell activation. The redistribution of spectrin coincides with the relocation of CD45 and LFA-1, two components essential for IS formation and stability. We assessed the role of spectrin by shRNA-mediated depletion from Jurkat T cells and show that spectrin-depleted cells exhibit decreased adhesion and are defective in forming lamellipodia and filopodia. Importantly, IS formation is impaired in spectrin-depleted cells. Thus, spectrin may be engaged in regulation of distinct events necessary for the establishment and maturity of the IS: besides the involvement of spectrin in the control of CD45 and LFA-1 surface display, spectrin acts in the establishment of cell-cell contact and adhesion processes during the formation of the IS.

Modeling of the axon membrane skeleton structure and implications for its mechanical properties.

Super-resolution microscopy recently revealed that, unlike the soma and dendrites, the axon membrane skeleton is structured as a series of actin rings connected by spectrin filaments that are held under tension. Currently, the structure-function relationship of the axonal structure is unclear. Here, we used atomic force microscopy (AFM) to show that the stiffness of the axon plasma membrane is significantly higher than the stiffnesses of dendrites and somata. To examine whether the structure of the axon plasma membrane determines its overall stiffness, we introduced a coarse-grain molecular dynamics model of the axon membrane skeleton that reproduces the structure identified by super-resolution microscopy. Our proposed computational model accurately simulates the median value of the Young's modulus of the axon plasma membrane determined by atomic force microscopy. It also predicts that because the spectrin filaments are under entropic tension, the thermal random motion of the voltage-gated sodium channels (Nav), which are bound to ankyrin particles, a critical axonal protein, is reduced compared to the thermal motion when spectrin filaments are held at equilibrium. Lastly, our model predicts that because spectrin filaments are under tension, any axonal injuries that lacerate spectrin filaments will likely lead to a permanent disruption of the membrane skeleton due to the inability of spectrin filaments to spontaneously form their initial under-tension configuration.

Nuclear mechanical resilience but not stiffness is modulated by αII-spectrin.

Spectrins are multi-domain, elastic proteins that provide elasticity to the plasma membrane of erythrocytes and select nucleated cells. Spectrins have also been found in the nucleus of non-erythrocytes, but their function remains to be uncovered. It has been hypothesized that a spring-like spectrin network exists within the lamina nucleoskeleton, however, experiments testing a spectrin network׳s mechanical impact on the nucleus are lacking. Here, we knock-down levels of nuclear αII-spectrin with the goal of disrupting this nucleoskeletal spectrin network. We mechanically test live cells with intranuclear particle tracking and compression assays to probe changes in nuclear mechanics with decreases in αII-spectrin. We show no changes in chromatin mechanics or in the stiffness of nuclei under compression. However, we do observe a reduction in the ability of nuclei with decreased αII-spectrin to recover after compression. These results establish spectrin as a nucleoskeletal component that specifically contributes to elastic recovery after compression.

Nuclear alpha spectrin: Critical roles in DNA interstrand cross-link repair and genomic stability.

Non-erythroid alpha spectrin (αIISp) is a structural protein which we have shown is present in the nucleus of human cells. It interacts with a number of nuclear proteins such as actin, lamin, emerin, chromatin remodeling factors, and DNA repair proteins. αIISp's interaction with DNA repair proteins has been extensively studied. We have demonstrated that nuclear αIISp is critical in DNA interstrand cross-link (ICL) repair in S phase, in both genomic (non-telomeric) and telomeric DNA, and in maintenance of genomic stability following ICL damage to DNA. We have proposed that αIISp acts as a scaffold aiding to recruit repair proteins to sites of damage. This involvement of αIISp in ICL repair and telomere maintenance after ICL damage represents new and critical functions for αIISp. These studies have led to development of a model for the role of αIISp in DNA ICL repair. They have been aided by examination of cells from patients with Fanconi anemia (FA), a repair-deficient genetic disorder in which a deficiency in αIISp leads to defective ICL repair in genomic and telomeric DNA, telomere dysfunction, and chromosome instability following DNA ICL damage. We have shown that loss of αIISp in FA cells is due to increased breakdown by the protease, µ-calpain. Importantly, we have demonstrated that this deficiency can be corrected by knockdown of µ-calpain and restoring αIISp levels to normal. This corrects a number of the phenotypic deficiencies in FA after ICL damage. These studies suggest a new and unexplored direction for therapeutically restoring genomic stability in FA cells and for correcting numerous phenotypic deficiencies occurring after ICL damage. Developing a more in-depth understanding of the importance of the interaction of αIISp with other nuclear proteins could significantly enhance our knowledge of the consequences of loss of αIISp on critical nuclear processes.

α-Spectrin and integrins act together to regulate actomyosin and columnarization, and to maintain a monolayered follicular epithelium.

The spectrin cytoskeleton crosslinks actin to the membrane, and although it has been greatly studied in erythrocytes, much is unknown about its function in epithelia. We have studied the role of spectrins during epithelia morphogenesis using the Drosophila follicular epithelium (FE). As previously described, we show that α-Spectrin and ß-Spectrin are essential to maintain a monolayered FE, but, contrary to previous work, spectrins are not required to control proliferation. Furthermore, spectrin mutant cells show differentiation and polarity defects only in the ectopic layers of stratified epithelia, similar to integrin mutants. Our results identify α-Spectrin and integrins as novel regulators of apical constriction-independent cell elongation, as α-Spectrin and integrin mutant cells fail to columnarize. Finally, we show that increasing and reducing the activity of the Rho1-Myosin II pathway enhances and decreases multilayering of α-Spectrin cells, respectively. Similarly, higher Myosin II activity enhances the integrin multilayering phenotype. This work identifies a primary role for α-Spectrin in controlling cell shape, perhaps by modulating actomyosin. In summary, we suggest that a functional spectrin-integrin complex is essential to balance adequate forces, in order to maintain a monolayered epithelium.

Caspase-3/7-mediated Cleavage of ß2-spectrin is Required for Acetaminophen-induced Liver Damage.

UNLABELLED: The ubiquitously expressed ß2-spectrin (ß2SP, SPTBN1) is the most common non-erythrocytic member of the ß-spectrin gene family. Loss of ß2-spectrin leads to defects in liver development, and its haploinsufficiency spontaneously leads to chronic liver disease and the eventual development of hepatocellular cancer. However, the specific role of ß2-spectrin in liver homeostasis remains to be elucidated. Here, we reported that ß2-spectrin was cleaved by caspase-3/7 upon treatment with acetaminophen which is the main cause of acute liver injury. Blockage of ß2-spectrin cleavage robustly attenuated ß2-spectrin-specific functions, including regulation of the cell cycle, apoptosis, and transcription. Cleaved fragments of ß2-spectrin were physiologically active, and the N- and C-terminal fragments retained discrete interaction partners and activity in transcriptional regulation and apoptosis, respectively. Cleavage of ß2-spectrin facilitated the redistribution of the resulting fragments under conditions of liver damage induced by acetaminophen. In contrast, downregulation of ß2-spectrin led to resistance to acetaminophen-induced cytotoxicity, and its insufficiency in the liver promoted suppression of acetaminophen-induced liver damage and enhancement of liver regeneration. CONCLUSIONS: ß2-Spectrin, a TGF-ß mediator and signaling molecule, is cleaved and activated by caspase-3/7, consequently enhancing apoptosis and transcriptional control to determine cell fate upon liver damage. These findings have extended our knowledge on the spectrum of ß2-spectrin functions from a scaffolding protein to a target and transmitter of TGF-ß in liver damage.

Cerebellar ataxias: ß-III spectrin's interactions suggest common pathogenic pathways.

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of disorders all characterised by postural abnormalities, motor deficits and cerebellar degeneration. Animal and in vitro models have revealed ß-III spectrin, a cytoskeletal protein present throughout the soma and dendritic tree of cerebellar Purkinje cells, to be required for the maintenance of dendritic architecture and for the trafficking and/or stabilisation of several membrane proteins: ankyrin-R, cell adhesion molecules, metabotropic glutamate receptor-1 (mGluR1), voltage-gated sodium channels (Nav ) and glutamate transporters. This scaffold of interactions connects ß-III spectrin to a wide variety of proteins implicated in the pathology of many SCAs. Heterozygous mutations in the gene encoding ß-III spectrin (SPTBN2) underlie SCA type-5 whereas homozygous mutations cause spectrin associated autosomal recessive ataxia type-1 (SPARCA1), an infantile form of ataxia with cognitive impairment. Loss-of ß-III spectrin function appears to underpin cerebellar dysfunction and degeneration in both diseases resulting in thinner dendrites, excessive dendritic protrusion with loss of planarity, reduced resurgent sodium currents and abnormal glutamatergic neurotransmission. The initial physiological consequences are a decrease in spontaneous activity and excessive excitation, likely to be offsetting each other, but eventually hyperexcitability gives rise to dark cell degeneration and reduced cerebellar output. Similar molecular mechanisms have been implicated for SCA1, 2, 3, 7, 13, 14, 19, 22, 27 and 28, highlighting alterations to intrinsic Purkinje cell activity, dendritic architecture and glutamatergic transmission as possible common mechanisms downstream of various loss-of-function primary genetic defects. A key question for future research is whether similar mechanisms underlie progressive cerebellar decline in normal ageing.

Posterior cerebellar Purkinje cells in an SCA5/SPARCA1 mouse model are especially vulnerable to the synergistic effect of loss of ß-III spectrin and GLAST.

Clinical phenotypes of spinocerebellar ataxia type-5 (SCA5) and spectrin-associated autosomal recessive cerebellar ataxia type-1 (SPARCA1) are mirrored in mice lacking ß-III spectrin (ß-III-/-). One function of ß-III spectrin is the stabilization of the Purkinje cell-specific glutamate transporter EAAT4 at the plasma membrane. In ß-III-/- mice EAAT4 levels are reduced from an early age. In contrast levels of the predominant cerebellar glutamate transporter GLAST, expressed in Bergmann glia, only fall progressively from 3 months onwards. Here we elucidated the roles of these two glutamate transporters in cerebellar pathogenesis mediated through loss of ß-III spectrin function by studying EAAT4 and GLAST knockout mice as well as crosses of both with ß-III-/- mice. Our data demonstrate that EAAT4 loss, but not abnormal AMPA receptor composition, in young ß-III-/- mice underlies early Purkinje cell hyper-excitability and that subsequent loss of GLAST, superimposed on the earlier deficiency of EAAT4, is responsible for Purkinje cell loss and progression of motor deficits. Yet the loss of GLAST appears to be independent of EAAT4 loss, highlighting that other aspects of Purkinje cell dysfunction underpin the pathogenic loss of GLAST. Finally, our results demonstrate that Purkinje cells in the posterior cerebellum of ß-III-/- mice are most susceptible to the combined loss of EAAT4 and GLAST, with degeneration of proximal dendrites, the site of climbing fibre innervation, most pronounced. This highlights the necessity for efficient glutamate clearance from these regions and identifies dysregulation of glutamatergic neurotransmission particularly within the posterior cerebellum as a key mechanism in SCA5 and SPARCA1 pathogenesis.

Anatomy of the red cell membrane skeleton: unanswered questions.

The red cell membrane skeleton is a pseudohexagonal meshwork of spectrin, actin, protein 4.1R, ankyrin, and actin-associated proteins that laminates the inner membrane surface and attaches to the overlying lipid bilayer via band 3-containing multiprotein complexes at the ankyrin- and actin-binding ends of spectrin. The membrane skeleton strengthens the lipid bilayer and endows the membrane with the durability and flexibility to survive in the circulation. In the 36 years since the first primitive model of the red cell skeleton was proposed, many additional proteins have been discovered, and their structures and interactions have been defined. However, almost nothing is known of the skeleton's physiology, and myriad questions about its structure remain, including questions concerning the structure of spectrin in situ, the way spectrin and other proteins bind to actin, how the membrane is assembled, the dynamics of the skeleton when the membrane is deformed or perturbed by parasites, the role lipids play, and variations in membrane structure in unique regions like lipid rafts. This knowledge is important because the red cell membrane skeleton is the model for spectrin-based membrane skeletons in all cells, and because defects in the red cell membrane skeleton underlie multiple hemolytic anemias.