Subcortical Cytoskeleton Research Articles

Raman spectroscopy-based approach to study the female gamete

In the last years, an increasing interest has emerged on the development of new non-invasive methods for the assessment of oocyte quality in order to improve outcomes of assisted reproductive technologies (ARTs) either in medical or veterinary fields.Raman microspectroscopy (RMS) has been proposed as a promising tool for the examination of the mammalian female gamete and identification of markers of its developmental competence. This technique provides a unique spectral fingerprint indicative of molecular composition of the cell and allows probing subcellular compartments. Studies have been carried out analysing by RMS fixed or living oocytes derived from different animal models. RMS imaging has been successfully applied to discriminate the biochemical changes of the global molecular architecture of mouse oocytes at different stages of maturation and those occurring in different conditions of maturation and oocyte aging. RMS can also detect modifications of specific structural components, including the oocyte zona pellucida and F-actin subcortical cytoskeleton in fresh sheep oocytes and those underwent to vitrification procedures. Finally, the recent application of Coherent anti-Stokes Raman scattering (CARS) microscopy for examination of oocyte lipid component will be briefly discussed. CARS overcomes some limits of RMS providing vibrational and spectral information with higher sensitivity, spatial resolution which is ideal to study living oocytes. This review summarizes the research on RMS approaches for oocyte evaluation showing the high potential use, current limitations and new improvements.

Structure and function of subcortical periodic cytoskeleton throughout the nervous system

Cytoskeleton plays an essential role in many functions in different cells and has been involved in the pathogenesis of many neural diseases. With the development of super-resolution fluorescence imaging technologies, which combine the molecular specificity and simple sample preparation of fluorescence microscopy and provide a spatial resolution comparable to that of electron microscopy, numerous new features have been revealed in the cytoskeletal organization of the subcortical cytoskeleton. A novel periodic lattice cytoskeleton is prevalent in different cell types throughout the nervous system. Here, we review the current studies of the molecular distribution, developmental mechanisms, and functional properties of the periodic cytoskeleton structure.

The Actin-Binding Protein α-Adducin Is Required for Maintaining Axon Diameter.

The actin-binding protein adducin was recently identified as a component of the neuronal subcortical cytoskeleton. Here, we analyzed mice lacking adducin to uncover the function of this protein in actin rings. α-adducin knockout mice presented progressive axon enlargement in the spinal cord and optic and sciatic nerves, followed by axon degeneration and loss. Using stimulated emission depletion super-resolution microscopy, we show that a periodic subcortical actin cytoskeleton is assembled in every neuron type inspected including retinal ganglion cells and dorsal root ganglia neurons. In neurons devoid of adducin, the actin ring diameter increased, although the inter-ring periodicity was maintained. In vitro, the actin ring diameter adjusted as axons grew, suggesting the lattice is dynamic. Our data support a model in which adducin activity is not essential for actin ring assembly and periodicity but is necessary to control the diameter of both actin rings and axons and actin filament growth within rings.

Subcortical cytoskeleton periodicity throughout the nervous system.

Superresolution fluorescence microscopy recently revealed a ~190 nm periodic cytoskeleton lattice consisting of actin, spectrin, and other proteins underneath the membrane of cultured hippocampal neurons. Whether the periodic cytoskeleton lattice is a structural feature of all neurons and how it is modified when axons are ensheathed by myelin forming glial cells is not known. Here, STED nanoscopy is used to demonstrate that this structure is a commonplace of virtually all neuron types in vitro. To check how the subcortical meshwork is modified during myelination, we studied sciatic nerve fibers from adult mice. Periodicity of both actin and spectrin was uncovered at the internodes, indicating no substantial differences between unmyelinated and myelinated axons. Remarkably, the actin/spectrin pattern was also detected in glial cells such as cultured oligodendrocyte precursor cells. Altogether our work shows that the periodic subcortical cytoskeletal meshwork is a fundamental characteristic of cells in the nervous system and is not a distinctive feature of neurons, as previously thought.

Loss of ezrin in human intrahepatic cholangiocarcinoma is associated with ectopic expression of E-cadherin

Ezrin connects proteins from the plasma membrane to the subcortical cytoskeleton, and contributes to epithelial integrity by interacting with the cell-cell adhesion molecule E-cadherin. In the liver, ezrin is restricted to cholangiocytes, where it regulates biliary secretory functions. During carcinogenesis, ezrin expression is impaired and associated with enhancement of cell migratory activity in cancer cells; therefore, we aimed to analyse ezrin in cholangiocarcinogenesis. Ezrin expression was evaluated by immunohistochemistry on tissue microarrays from 94 surgical specimens of intrahepatic cholangiocarcinoma (CCA), and correlated with clinicopathological factors and E-cadherin expression. Ezrin function was also analysed in human CCA cell lines. In CCA, ezrin was negative/weakly expressed in 49 cases (52%) and moderately/strongly expressed in 45 cases (48%), mostly in cell cytoplasm. The negative/weak expression of ezrin was more frequent in peripheral than in perihilar CCA (P = 0.002), and was associated with high tumour size (P = 0.001), low mucus secretion (P = 0.042), the presence of satellite nodules (P = 0.024), and ectopic cytoplasmic expression of E-cadherin (P = 0.005). In vitro, silencing of ezrin in CCA cells caused internalization of E-cadherin and favoured cell migration. Ezrin is down-regulated during cholangiocarcinogenesis, and its loss results in a more aggressive phenotype.

Molecular handoffs in nitrergic neurotransmission.

Postsynaptic density (PSD) proteins in excitatory synapses are relatively immobile components, while there is a structured organization of mobile scaffolding proteins lying beneath the PSDs. For example, shank proteins are located further away from the membrane in the cytosolic faces of the PSDs, facing the actin cytoskeleton. The rationale of this organization may be related to important roles of these proteins as “exchange hubs” for the signaling proteins for their migration from the subcortical cytosol to the membrane. Notably, PSD95 have also been demonstrated in prejunctional nerve terminals of nitrergic neuronal varicosities traversing the gastrointestinal smooth muscles. It has been recently reported that motor proteins like myosin Va play important role in transcytosis of nNOS. In this review, the hypothesis is forwarded that nNOS delivered to subcortical cytoskeleton requires interactions with scaffolding proteins prior to docking at the membrane. This may involve significant role of “shank,” named for SRC-homology (SH3) and multiple ankyrin repeat domains, in nitric oxide synthesis. Dynein light chain LC8–nNOS from acto-myosin Va is possibly exchanged with shank, which thereafter facilitates transposition of nNOS for binding with palmitoyl-PSD95 at the nerve terminal membrane. Shank knockout mice, which present with features of autism spectrum disorders, may help delineate the role of shank in enteric nitrergic neuromuscular transmission. Deletion of shank3 in humans is a monogenic cause of autism called Phelan–McDermid syndrome. One fourth of these patients present with cyclical vomiting, which may be explained by junctionopathy resulting from shank deficit in enteric nitrergic nerve terminals.

Increased Cytoskeletal Stiffness of Schlemm's Canal Endothelial Cells in Glaucoma

The elevated intraocular pressure characteristic of glaucoma has been associated with a decreased pore density in the cells of the inner wall of Schlemm's canal (SC). SC cells form these pores in response to a transcellular pressure drop, facilitating the flow of aqueous humor across this endothelium. We hypothesize that impaired pore formation in glaucoma is due to an elevated stiffness of SC cells.Atomic force microscopy (AFM) measurements of elastic modulus were performed using pyramidal or spherical (4.5 or 10 µm) tips on SC cells isolated from 6 healthy and 5 glaucomatous human eyes. Using finite element modeling and AFM experiments with latrunculin-A (an F-actin depolymerizing agent), we previously showed that sharp pyramidal tips characterize the cortex stiffness while larger, spherical tips characterize the stiffness of the subcortical cytoskeleton. The geometry of an SC cell was reconstructed based on electron microscopy images and used to model cell deformation under pressure (3-6 mmHg).When probed with spherical tips, the modulus of glaucomatous cells (1.36±0.14 kPa) was significantly higher (p<0.02) than that of healthy cells (0.89±0.10 kPa). No significant difference was detected between healthy and glaucomatous cells using sharp tips (7.40±1.33 vs. 7.99±0.97 kPa). The higher modulus measured on glaucomatous cells using spherical tips suggest that the altered stiffness is likely in the subcortical cytoskeleton and not in the cell cortex. Preliminary studies using finite element modeling predict a 34-40% decrease in cell deformation solely due to the increased cell stiffness measured on glaucomatous cells. This is consistent with our hypothesis that cell deformation is likely a precursor to the pressure-driven pore formation process and that increased cell stiffness may inhibit this process.Acknowledgements: BrightFocus Foundation, NIH EY019696, NIH T32 EY007128.

Systems Genetics Implicates Cytoskeletal Genes in Oocyte Control of Cloned Embryo Quality

Cloning by somatic cell nuclear transfer is an important technology, but remains limited due to poor rates of success. Identifying genes supporting clone development would enhance our understanding of basic embryology, improve applications of the technology, support greater understanding of establishing pluripotent stem cells, and provide new insight into clinically important determinants of oocyte quality. For the first time, a systems genetics approach was taken to discover genes contributing to the ability of an oocyte to support early cloned embryo development. This identified a primary locus on mouse chromosome 17 and potential loci on chromosomes 1 and 4. A combination of oocyte transcriptome profiling data, expression correlation analysis, and functional and network analyses yielded a short list of likely candidate genes in two categories. The major category-including two genes with the strongest genetic associations with the traits (Epb4.1l3 and Dlgap1)-encodes proteins associated with the subcortical cytoskeleton and other cytoskeletal elements such as the spindle. The second category encodes chromatin and transcription regulators (Runx1t1, Smchd1, and Chd7). Smchd1 promotes X chromosome inactivation, whereas Chd7 regulates expression of pluripotency genes. Runx1t1 has not been associated with these processes, but acts as a transcriptional repressor. The finding that cytoskeleton-associated proteins may be key determinants of early clone development highlights potential roles for cytoplasmic components of the oocyte in supporting nuclear reprogramming. The transcriptional regulators identified may contribute to the overall process as downstream effectors.

CFTR regulation in human airway epithelial cells requires integrity of the actin cytoskeleton and compartmentalized cAMP and PKA activity

The cystic fibrosis transmembrane conductance regulator (CFTR) mutation ΔF508CFTR still causes regulatory defects when rescued to the apical membrane, suggesting that the intracellular milieu might affect its ability to respond to cAMP regulation. We recently reported that overexpression of the Na(+)/H(+) exchanger regulatory factor NHERF1 in the cystic fibrosis (CF) airway cell line CFBE41o-rescues the functional expression of ΔF508CFTR by promoting F-actin organization and formation of the NHERF1-ezrin-actin complex. Here, using real-time FRET reporters of both PKA activity and cAMP levels, we find that lack of an organized subcortical cytoskeleton in CFBE41o-cells causes both defective accumulation of cAMP in the subcortical compartment and excessive cytosolic accumulation of cAMP. This results in reduced subcortical levels and increased cytosolic levels of PKA activity. NHERF1 overexpression in CFBE41o-cells restores chloride secretion, subcortical cAMP compartmentalization and local PKA activity, indicating that regulation of ΔF508CFTR function requires not only stable expression of the mutant CFTR at the cell surface but also depends on both generation of local cAMP signals of adequate amplitude and activation of PKA in proximity of its target. Moreover, we found that the knockdown of wild-type CFTR in the non-CF 16HBE14o-cells results in both altered cytoskeletal organization and loss of cAMP compartmentalization, whereas stable overexpression of wt CFTR in CF cells restores cytoskeleton organization and re-establishes the compartmentalization of cAMP at the plasma membrane. This suggests that the presence of CFTR on the plasma membrane influences the cytoskeletal organizational state and, consequently, cAMP distribution. Our data show that a sufficiently high concentration of cAMP in the subcortical compartment is required to achieve PKA-mediated regulation of CFTR activity.

The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor β-Stimulated Aortic Endothelial Cells

Podosomes are dynamic actin-rich adhesion plasma membrane microdomains endowed with extracellular matrix-degrading activities. In aortic endothelial cells, podosomes are induced by transforming growth factor β (TGF-β), but how this occurs is largely unknown. It is thought that, in endothelial cells, podosomes play a role in vessel remodeling and/or in breaching anatomical barriers. We demonstrate here that, in bovine aortic endothelial cells, that the Cdc42-specific guanine exchange factor (GEF) Fgd1 is expressed and regulated by TGF-β to induce Cdc42-dependent podosome assembly. Within 15 min of TGF-β stimulation, Fgd1, but none of the other tested Cdc42 GEFs, undergoes tyrosine phosphorylation, associates with Cdc42, and translocates to the subcortical cytoskeleton via a cortactin-dependent mechanism. Small interfering RNA-mediated Fgd1 knockdown inhibits TGF-β-induced Cdc42 activation. Fgd1 depletion also reduces podosome formation and associated matrix degradation and these defects are rescued by reexpression of Fgd1. Although overexpression of Fgd1 does not promote podosome formation per se, it enhances TGF-β-induced matrix degradation. Our results identify Fgd1 as a TGF-β-regulated GEF and, as such, the first GEF to be involved in the process of cytokine-induced podosome formation. Our findings reveal the involvement of Fgd1 in endothelial cell biology and open up new avenues to study its role in vascular pathophysiology.

Line tension at lipid phase boundaries regulates formation of membrane vesicles in living cells

Ternary lipid compositions in model membranes segregate into large-scale liquid-ordered (L o) and liquid-disordered (L d) phases. Here, we show μm-sized lipid domain separation leading to vesicle formation in unperturbed human HaCaT keratinocytes. Budding vesicles in the apical portion of the plasma membrane were predominantly labelled with L d markers 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate and weakly stained by L o marker fluorescein-labeled cholera toxin B subunit which labels ganglioside GM 1 enriched plasma membrane rafts. Cholesterol depletion with methyl-β-cyclodextrin enhanced DiI vesiculation, GM 1/DiI domain separation and was accompanied by a detachment of the subcortical cytoskeleton from the plasma membrane. Based on these observations we describe the energetic requirements for plasma membrane vesiculation. We propose that the decrease in total ‘L o/L d’ boundary line tension arising from the coalescence of smaller L d-like domains makes it energetically favourable for L d-like domains to bend from flat μm-sized surfaces to cap-like budding vesicles. Thus living cells may utilize membrane line tension energies as a control mechanism of exocytic events.

Drosophila non-muscle α-actinin is localized in nurse cell actin bundles and ring canals, but is not required for fertility

The single copy Drosophila α-actinin gene is alternatively spliced to generate three different isoforms that are expressed in larval muscle, adult muscle and non-muscle cells, respectively. We have generated novel α-actinin alleles, which specifically remove the non-muscle isoform. Homozygous mutant flies are viable and fertile with no obvious defects. Using a monoclonal antibody that recognizes all three splice variants, we compared α-actinin distribution in wild type and mutant embryos and ovaries. We found that non-muscle α-actinin was present in young embryos and in the embryonic central nervous system. In ovaries, non-muscle α-actinin was localized in the nurse cell subcortical cytoskeleton, cytoplasmic actin cables and ring canals. In the mutant, α-actinin expression remained in muscle tissues, but also in a subpopulation of epithelial cells in both embryos and ovaries. This suggests that various populations of non-muscle cells regulate α-actinin expression in different ways. We also show that ectopically expressed adult muscle-specific α-actinin localizes to all F-actin containing structures in the nurse cells in the absence of endogenous non-muscle α-actinin.

G Proteins, Plant Growth, and Parasite Invasion

The function of regulators of G protein signaling (RGSs) is just as the name indicates--in mammals and yeast, these proteins attenuate signals initiated by G protein-coupled receptors (GPCRs) at the cell surface. Chen et al. have now identified an RGS protein in Arabidopsis that functions together with a heterotrimeric G protein to control root and shoot growth. The plant RGS behaves biochemically like a bona fide family member, yet its sequence predicts a protein that contains a seven-transmembrane spanning region similar to GPCRs, quite unlike other known RGS proteins. The process of invasion of red blood cells by the malaria parasite Plasmodium falciparum is fundamental to disease progression. Harrison et al. reveal the requirement for GPCR activation in the host erythrocyte cell membrane to allow successful invasion. It is likely that the activated receptors promote the rearrangement of the rigid erythrocyte subcortical cytoskeleton to allow for the deformation of the membrane required during parasite invasion. J.-G. Chen, F. S. Willard, J. Huang, J. Liang, S. A. Chasse, A. M. Jones, D. P. Siderovski, A seven-transmembrane RGS protein that modulates plant cell proliferation. Science 301 , 1728-1731 (2003). [Abstract] [Full Text] T. Harrison, B. U. Samuel, T. Akompong, H. Hamm, N. Mohandas, J. W. Lomasney, K. Haldar, Erythrocyte G protein-coupled receptor signaling in malarial infection. Science 301 , 1734-1736 (2003). [Abstract] [Full Text]

Mechanical properties of alveolar epithelial cells in culture.

With the use of magnetic twisting cytometry, we characterized the mechanical properties of rat type II alveolar epithelial (ATII) cells in primary culture and examined whether the cells' state of differentiation and the application of deforming stresses influence their resistance to shape change. Cells were harvested from rat lungs as previously described (Dobbs LG. Am J Physiol Lung Cell Mol Physiol 258: L134-L147, 1990) and plated at a density of 1 x 10(6) cells/cm(2) in fibronectin-coated 96 Remova wells, and their mechanical properties were measured 2-9 days later. We show 1) that ATII cells form much stronger bonds with RGD-coated beads than they do with albumin- or acetylated low-density lipoprotein-coated beads, 2) that RGD-mediated bonds seemingly "mature" during the first 60 min of bead contact, 3) that the apparent stiffness of ATII cells increases with days in culture, 4) that stiffness falls when the RGD-coated beads are intermittently oscillated at 0.3 Hz, and 5) that this fall cannot be attributed to exocytosis-related remodeling of the subcortical cytoskeleton. Although the mechanisms of force transfer between basement membrane, cytoskeleton, and plasma membrane of ATII cells remain to be resolved, such analyses undoubtedly require definition of the cell's mechanical properties. To our knowledge, the results presented here provide the first data on this topic.

Microbes and microbial toxins: paradigms for microbial-mucosal interactions III. Shigellosis: from symptoms to molecular pathogenesis.

Interaction of Shigella flexneri with epithelial cells includes contact of bacteria with the cell surface and release of Ipa proteins through a specialized type III secreton. A complex signaling process involving activation of small GTPases of the Rho family and c-src causes major rearrangements of the subcortical cytoskeleton, thereby allowing bacterial entry by macropinocytosis. After entry, shigellae escape to the cell cytoplasm and initiate intracytoplasmic movement through polar nucleation and assembly of actin filaments caused by bacterial surface protein IcsA, which binds and activates neuronal Wiskoff-Aldrich syndrome protein (N-WASP), thus inducing actin nucleation in an Arp 2/3-dependent mechanism. Actin-driven motility promotes efficient colonization of the host cell cytoplasm and rapid cell-to-cell spread via protrusions that are engulfed by adjacent cells in a cadherin-dependent process. Bacterial invasion turns infected cells to strongly proinflammatory cells through sustained activation of nuclear factor-kappaB. A major consequence is interleukin (IL)-8 production, which attracts polymorphonuclear leukocytes (PMNs). On transmigration, PMNs disrupt the permeability of this epithelium and promote its invasion by shigellae. At the early stage of infection, M cells of the follicle-associated epithelium allow bacterial translocation. Subsequent apoptotic killing of macrophages in a caspase 1-dependent process causes the release of IL-1beta and IL-18, which accounts for the initial steps of inflammation.

(Alpha)3(beta)1 integrin regulates epithelial cytoskeletal organization.

Epithelial cell morphology and cytoskeletal organization are determined by interactions, with both adjacent cells and the extracellular matrix, which are mediated by integrins and cadherins. Little is known, however, of the relative contributions of integrins and cadherins to maintaining the sub-cortical cytoskeleton characteristic of epithelial cells. Since most studies that utilize integrin-blocking antibodies result in a loss of both cell-cell adhesion and sub-cortical cytoskeletal organization, it has been difficult to distinguish whether integrins and cadherins both mediate cytoskeletal assembly in epithelial cells. Therefore, cells derived from kidney collecting ducts of (alpha)3(beta)1 integrin-deficient mice were used to examine the role of integrins in epithelial cell morphology and cytoskeletal organization. In primary cell culture, (alpha)3(beta)1 integrin-deficient kidney collecting duct cells maintain cadherin-mediated cell-cell adhesions but fail to form the sub-cortical cytoskeleton that is characteristic of epithelial cells, and instead assemble actin stress fibers. Moreover, the cell-cell junctions in mutant cells were irregular, rather than being uniformly oriented perpendicular to the culture substrate. These results demonstrated that integrins have an primary and essential function in establishing and maintaining the sub-cortical cytoskeleton that is characteristic of epithelial cells. To further study the role of (alpha)3(beta)1 integrin in establishing and maintaining cytoskeletal organization in tubular epithelial cells, we derived immortalized cell lines from wild-type and (alpha)3(beta)1 integrin-deficient kidney collecting ducts that duplicated the cytoskeletal and cadherin organization observed in primary cells. E-cadherin and (alpha)- and (beta)-catenin were complexed together in equal amounts in membranes of wild-type and (alpha)3(beta)1 integrin-deficient cells. However, association of the cadherin:catenin complex with (alpha)-actinin was greatly decreased in mutant cells, indicating that integrin-mediated assembly of the sub-cortical cytoskeleton is essential for subsequent association of the cytoskeleton with the cadherin:catenin complex. These results present direct evidence for integrin:cadherin cross-regulation in which cadherin function is dependent on the presence of an integrin.

Evidence for the Participation of the Neuron-Specific CDK5 Activator P35 during Laminin-Enhanced Axonal Growth

Cultures of cerebellar macroneurons were used to study the pattern of expression, subcellular localization, and function of the neuronal cdk5 activator p35 during laminin-enhanced axonal growth. The results obtained indicate that laminin, an extracellular matrix molecule capable of selectively stimulating axonal extension and promoting MAP1B phosphorylation at a proline-directed protein kinase epitope, selectively stimulates p35 expression, increases its association with the subcortical cytoskeleton, and accelerates its redistribution to the axonal growth cones. Besides, suppression of p35, but not of a highly related isoform designated as p39, by antisense oligonucleotide treatment selectively reduces cdk5 activity, laminin-enhanced axonal elongation, and MAP1b phosphorylation. Taken collectively, the present results suggest that cdk5/p35 may serve as an important regulatory linker between environmental signals (e.g., laminin) and constituents of the intracellular machinery (e.g., MAP1B) involved in axonal elongation.

Analysis of the expression, distribution and function of cyclin dependent kinase 5 (cdk5) in developing cerebellar macroneurons.

Cultures of cerebellar macroneurons were used to study the expression, activity, subcellular localization, and function of cdk5 during neuronal morphogenesis. The results obtained indicate that in non-polarized neurons cdk5 is restricted to the cell body but as soon as polarity is established it becomes highly concentrated at the distal tip of growing axons where it associates with microtubules and the subcortical cytoskeleton. In addition, we show that laminin, an extracellular matrix molecule capable of stimulating axonal extension and promoting MAP1b phosphorylation (DiTella et al., 1996), accelerates the redistribution of cdk5 to the axonal tip and dramatically increases its activity. Finally, our results indicate that cdk5 suppression by antisense oligonucleotide treatment selectively reduces axonal elongation and decreases the phosphorylation status of MAP1b, as well as its binding to microtubules. Taken collectively, our observations suggest that cdk5 may serve as an important regulatory linker between environmental signals (e.g. laminin) and constituents of the intracellular machinery (e.g. MAP1b) involved in axonal formation.

Unraveling the cytoplasmic interactions of the cadherin superfamily.

Unraveling the cytoplasmic interactions of the cadherin superfamily.

Na+,K+-adenosine triphosphatase polarity in retinal photoreceptors: a role for cytoskeletal attachments.

We have used isolated embryonic photoreceptor cells as a model system with which to examine the mechanisms responsible for the development and maintenance of asymmetric Na+,K+-ATPase (ATPase) distribution. Photoreceptor precursors, which appear round and process free at culture onset, develop structural and molecular properties similar to those of photoreceptor cells in vivo. ATPase, recognized by an anti-ATPase antibody, is distributed over the entire surface of round photoreceptor precursors. As the cells develop, ATPase becomes progressively concentrated in the inner segment (where it is found in cells of the intact retina). This phenomenon occurs in cells developing in the absence of intercellular contacts. The development of ATPase polarity correlates with a decrease in the fraction of ATPase molecules that are mobile in the membrane (as determined by fluorescence photobleaching recovery), as well as with an increase in the fraction of ATPase that remains associated with the cells after detergent extraction. The magnitudes of the mobile ATPase fractions agree well with those of the detergent-extractable fractions in both the immature and developed photoreceptors. The distribution of alpha spectrin and ATPase-immunoreactive materials appeared qualitatively similar, and quantitative image analysis showed similar gradients of spectrin and Na+,K+-ATPase immunofluorescence along the long axis of elongated photoreceptors. Moreover, detergent extractability of alpha spectrin and the ATPase showed similar modifications in response to changes in pH or KCl concentration. ATPase detergent-extractable and mobile fractions were not changed in cultures treated with cytoskeletal inhibitors such as nocodazole. These data are consistent with a role for an asymmetrically distributed, spectrin-containing subcortical cytoskeleton in the preferential accumulation of Na+,K+-ATPase in the photoreceptor inner segment.