Amount Of F-actin Research Articles

Direct evidence for roles of phosphorylated regulatory light chain of myosin II in furrow ingression during cytokinesis in HeLa cells

Phosphorylation of myosin II is thought to play an important role in cytokinesis. Although it is well known that phosphorylated regulatory light chain of myosin II (P-MRLC) localizes along the contractile ring, it is not clear how P-MRLC controls myosin II and F-actin in furrow ingression during cytokinesis. To elucidate roles of P-MRLC in furrow ingression, HeLa cells transfected with EGFP-tagged wild-type or each MRLC mutant were observed using a live-imaging microscope. Time-lapse observation revealed that a delay of furrow ingression was observed in the nonphosphorylatable form of MRLC (AA-MRLC)-expressing cell but not in the wild-type or phospho-mimic MRLC-expressing cell. Among each form of MRLC-expressing cell, the total amount of P-MRLC including phospho-mimic MRLCs was smallest in the cell expressing AA-MRLC. However, the amount of F-actin and myosin II at the contractile ring in the AA-MRLC-expressing cell was the same as that in the normal cell. Interestingly, delay of furrow ingression by a Rho-kinase inhibitor, Y27632, was rescued by phospho-mimic MRLCs. These results suggest that the P-MRLC is essential for the progress of furrow ingression but not the retainment of F-actin and myosin II in the contractile ring of dividing HeLa cells.

Stress-regulated kinase pathways in the recovery of tip growth and microtubule dynamics following osmotic stress inS. pombe

The cell-integrity and stress-response MAP kinase pathways (CIP and SRP, respectively) are stimulated by various environmental stresses. Ssp1 kinase modulates actin dynamics and is rapidly recruited to the plasma membrane following osmotic stress. Here, we show that osmotic stress arrested tip growth, induced the deposition of abnormal cell-wall deposits at tips and led to disassociation of F-actin foci from cell tips together with a reduction in the amount of F-actin in these foci. Osmotic stress also ;froze' the dynamics of interphase microtubule bundles, with microtubules remaining static for approximately 38 minutes (at 30 degrees C) before fragmenting upon return to dynamic behaviour. The timing with which microtubules resumed dynamic behaviour relied upon SRP activation of Atf1-mediated transcription, but not on either CIP or Ssp1 signalling. Analysis of the recovery of tip growth showed that: (1) the timing of recovery was controlled by SRP-stimulated Atf1 transcription; (2) re-establishment of polarized tip growth was absolutely dependent upon SRP and partially dependent upon Ssp1 signalling; and (3) selection of the site for polarized tip extension required Ssp1 and the SRP-associated polarity factor Wsh3 (also known as Tea4). CIP signalling did not impact upon any aspect of recovery. The normal kinetics of tip growth following osmotic stress of plo1.S402A/E mutants established that SRP control over the resumption of tip growth after osmotic stress is distinct from its control of tip growth following heat or gravitational stresses.

Marine toxins and the cytoskeleton: okadaic acid and dinophysistoxins

Okadaic acid (OA) and its analogs, the dinophysistoxins, are potent inhibitors of protein phosphatases 1 and 2A. This action is well known to cause diarrhea and gastrointestinal symptons when the toxins reach the digestive tract by ingestion of mollusks. A less well-known effect of these group of toxins is their effect in the cytoskeleton. OA has been shown to stimulate cell motility, loss of stabilization of focal adhesions and a consequent loss of cytoskeletal organization due to an alteration in the tyrosine-phosphorylated state of the focal adhesion kinases and paxillin. OA causes cell rounding and loss of barrier properties through mechanisms that probably involve disruption of filamentous actin (F-actin) and/or hyperphosphorylation and activation of kinases that stimulate tight junction disassembly. Neither methyl okadaate (a weak phosphatase inhibitor) nor OA modify the total amount of F-actin, but both toxins cause similar changes in the F-actin cytoskeleton, with strong retraction and rounding, and in many cases cell detachment. OA and dinophysistoxin-1 (35S-methylokadaic acid) cause rapid changes in the structural organization of intermediate filaments, followed by a loss of microtubules, solubilization of intermediate filament proteins, and disruption of desmosomes. The detailed pathways that coordinate all these effects are not yet known.

Decreased tyrosine phosphorylation of nephrin in rat and human nephrosis

Phosphorylation of tyrosine residue (Y1204) of rat nephrin by Fyn kinase allows Nck adaptor protein binding to nephrin motifs, which include the phosphorylated tyrosine. This phosphorylation-dependent switch induces actin polymerization in a cell culture system. Here, we generated an antibody recognizing phosphorylated nephrin at the Nck binding sites pY1204 and pY1228 to determine the phosphorylation status of nephrin using a rat model of puromycin aminonucleoside-induced nephrosis. Changes in globular actin (G-actin) and filamentous actin (F-actin) contents in isolated glomeruli were measured by western blot. Before experimental nephrosis, both Y1204 and Y1228 were phosphorylated, and most of the actin was filamentous. Before the onset of overt proteinuria, however, phosphorylation of both Y1204 and Y1228 rapidly decreased and became almost undetectable. During this period, the amount of F-actin in glomeruli began to decrease, whereas G-actin increased. Phosphorylation of nephrin at Y1228 in glomeruli of patients with minimal change nephrosis was significantly decreased compared with that in normal glomeruli. Our study suggests that tyrosine phosphorylation of nephrin by regulating F-actin formation may be important for the maintenance of normal podocyte morphology and function.

MDia2 Induces the Actin Scaffold for the Contractile Ring and Stabilizes Its Position during Cytokinesis in NIH 3T3 Cells

mDia proteins are mammalian homologues of Drosophila diaphanous and belong to the formin family proteins that catalyze actin nucleation and polymerization. Although formin family proteins of nonmammalian species such as Drosophila diaphanous are essential in cytokinesis, whether and how mDia proteins function in cytokinesis remain unknown. Here we depleted each of the three mDia isoforms in NIH 3T3 cells by RNA interference and examined this issue. Depletion of mDia2 selectively increased the number of binucleate cells, which was corrected by coexpression of RNAi-resistant full-length mDia2. mDia2 accumulates in the cleavage furrow during anaphase to telophase, and concentrates in the midbody at the end of cytokinesis. Depletion of mDia2 induced contraction at aberrant sites of dividing cells, where contractile ring components such as RhoA, myosin, anillin, and phosphorylated ERM accumulated. Treatment with blebbistatin suppressed abnormal contraction, corrected localization of the above components, and revealed that the amount of F-actin at the equatorial region during anaphase/telophase was significantly decreased with mDia2 RNAi. These results demonstrate that mDia2 is essential in mammalian cell cytokinesis and that mDia2-induced F-actin forms a scaffold for the contractile ring and maintains its position in the middle of a dividing cell.

Induction of actin cytoskeleton rearrangement by methyl okadaate - comparison with okadaic acid

Methyl okadaate is a derivative of the lipophilic polyether okadaic acid (OA), a well-known inducer of apoptosis. OA inhibits Ser/Thr protein phosphatases (PPs), among them types 1 and 2A (PP1 and PP2A), whereas methyl okadaate lacks PP1/PP2A inhibitory activity in vitro. As progressive loss of neuronal cytoarchitecture is a major event that precedes neuronal death, in this work we studied comparatively the effects of both toxins on actin cytoskeleton organization in human neuroblastoma cells by filamentous actin (F-actin) labeling with the specific dye Oregon Green 514 Phalloidin. Neither methyl okadaate nor OA modified the amount of F-actin per cell. However, confocal microscopy imaging showed that methyl okadaate induced reorganization of actin cytoskeleton, loss of the typical flattened morphology and adoption of a round shape, and a reduction in the number of neurites, with a consequent loss of cell attachment. These effects were identical to those induced by OA, although methyl okadaate potency was approximately 10-fold lower. In order to investigate the role of membrane potential and cytosolic Ca2+ concentration in morphological changes induced by these toxins, the cells were stained with bis-(1,3-dibutylbarbituric acid)-trimethine oxonol and fura-2. No toxin effect was detected on membrane potential or calcium influx, indicating that these two signals are not responsible for cytoskeletal/morphological change induction. Methyl okadaate induced an increase of Ser/Thr phosphorylation of cellular proteins detected by western blot, showing similar phosphorylation profiles to OA. Our data suggest that methyl okadaate is an active compound that shares a pharmacological target with OA that may be a Ser/Thr phosphatase, probably different from PP1 and PP2A.

Biomechanical analysis of cancerous and normal cells based on bulge generation in a microfluidic device

This paper presents a new biomechanical analysis method for discrimination between cancerous and normal cells through compression by poly(dimethylsiloxane) (PDMS) membrane deflection in a microfluidic device. When a cell is compressed, cellular membrane will expand and then small bulges will appear on the peripheral cell membrane beyond the allowable strain. It is well known that the amount of F-actin in cancer cells is less than that of normal cells and bulges occur at the sites where cytoskeleton becomes detached from the membrane bilayer. Accordingly, we have demonstrated the difference of the bulge generation between breast cancer cells (MCF7) and normal cells (MCF10A). After excessive deformation, the bulges generated in MCF7 cells were not evenly distributed on the cell periphery. Contrary to this, the bulges of MCF10A cells showed an even distribution. In addition, the morphologies of bulges of MCF7 and MCF10A cells looked swollen protrusion and tubular protrusion, respectively. Peripheral strains at the moment of the bulge generation were also 72% in MCF7 and 46% in MCF10A. The results show that the bulge generation can be correlated with the cytoskeleton quantity inside the cell, providing the first step of a new biomechanical approach.

Cyclic stretching promotes collagen synthesis and affects F-actin distribution in rat mesenchymal stem cells

Recent studies have shown effects of mechanical environment on bone marrow mesenchymal stem cells (BMSC). In order to examine how BMSC and their cytoskeleton respond to mechanical stimulation, we investigated their collagen synthesis and F-actin expression. Rat BMSC were harvested from adult rats and cultured to passage 4. Then the cells were seeded onto a silicone membrane loaded with an uniaxial cyclic stretching (10%, 1 Hz) during 3, 6, 12, 24 and 36 h. The levels of collagen type I and III before and after stretching were analyzed by immunocytochemistry, and the F-actin in cytoplasm was observed by confocal microscopy. Immunocytochemistry results showed that the stretching enhanced the synthesis of collagen types I and III in BMSC after 24 h stimulation. However, a decrease in fluorescence density of F-actin was observed after the stretching in a time dependent manner. In addition, the F-actin filaments seemed much thinner than those of static cells. These results indicated that the cyclic stretching favored the synthesis of collagen types I and III, but decreased the amount of F-actin in the BMSC.

The Role of DNA and Actin Polymers on the Polymer Structure and Rheology of Cystic Fibrosis Sputum and Depolymerization by Gelsolin or Thymosin Beta 4

Mucus clearance is the first line of pulmonary defense against inhaled irritants, microorganisms, and allergens. In health, the gel-forming mucins are the principal polymeric components of airway mucus but in cystic fibrosis (CF), the necrotic death of inflammatory and epithelial cells releases a network of copolymerized extracellular DNA and filamentous (F-) actin-producing secretions that are similar to pus and difficult to clear by cilia or airflow. The large amounts of F-actin in CF sputum suggested that thymosin beta4 (Tbeta4) or gelsolin could depolymerize the secondary polymer network of CF sputum. Dose-dependent CF sputum rheology and polymer structure were measured before and after the addition of excipient, dornase alfa, Tbeta4, gelsolin, and Tbeta4 or gelsolin with dornase for 30 min. Sputum was also incubated with Tbeta4 30 microg/mL, gelsolin 10 microg/mL or excipient for 0, 5, 10, 15, 20, or 60 min. There was a dose-dependent decrease in cohesivity with Tbeta4 and a time-dependent decrease in cohesivity at 30 microg/mL. With the combination of dornase alfa and Tbeta4 at 1.5 microg/mL, there was a 65% decrease in elasticity (P = 0.013). There was a time-dependent decrease in cohesivity (P = 0.0004) and elasticity (P = 0.047) with gelsolin and a dose-dependent fall in cohesivity (P = 0.0008). An apparent synergy of Tbeta4 or gelsolin on actin and dornase on DNA may be explained by the combined effect of actin depolymerization and DNA filament severing or by virtue of actin depolymerization increasing the effectiveness of dornase alfa.

Effects of heat stress on filamentous actin and prestin of outer hair cells in mice

When the ear is exposed to traumatic loud noise, outer hair cells (OHCs) are damaged and thus permanent hearing loss occurs. Recently, prior conditioning with heat stress has been reported to protect OHCs from traumatic noise exposure by increasing the stiffness of the OHC soma and has also been reported to enhance distortion product otoacoustic emissions [DPOAEs; Murakoshi, M., Yoshida, N., Kitsunai, Y., Iida, K., Kumano, S., Suzuki, T., Kobayashi, T., Wada, H., 2006. Effects of heat stress on Young's modulus of outer hair cells in mice. Brain Res. 1107, 121–130]. In the present study, to further investigate the heat stress-induced protective mechanism of hearing and such stress-induced DPOAE enhancement mechanism, the amount of filamentous actin (F-actin), which is concerned with cell stiffness, and the amount of prestin, which is concerned with the generation of DPOAEs, were examined in OHCs, with and without heat stress. Heat stress was found to increase the amount of F-actin 6–24 h after heat stress. The greatest increase in the amount of F-actin was observed at the cuticular plate where F-actin anchors the roots of the stereocilia to the cell body. Based on this result, the part of the stereocilia most reinforced and protected by heat stress was concluded to be the roots of the stereocilia. In contrast with F-actin, heat stress did not affect the amount of prestin.

Control of Actin Assembly Dynamics in Cell Motility

Control of Actin Assembly Dynamics in Cell Motility

Ischemic Preconditioning and Intermittent Ischemia Preserve Bile Flow in a Rat Model of Ischemia/Reperfusion Injury

Ischemia and reperfusion (IR) injury of the liver is associated with impaired bile secretion, but the effects of ischemic preconditioning (IPC) and intermittent ischemia (INT) on bile flow are unknown. A rat model of segmental (60%-70%) hepatic ischemia and reperfusion was employed to test the effects of IPC and INT on bile flow. Continuous clamping for 45 min (CC) substantially reduced bile flow, and this did not recover after 60 min of reperfusion. IPC and INT caused a significant recovery of bile flow. The elevation in plasma liver marker enzymes induced by CC was not reduced by IPC and INT. Light microscopy showed mild hepatocyte damage in all groups. In the CC group, the amount of F-actin localized around the bile canaliculi in the ischemic lobes was less than that in the nonischemic lobes, but this difference was not observed in the IPC and INT groups. It is concluded that IPC and INT substantially alleviate the decrease in bile flow induced by ischemia. Bile flow may be useful in the assessment of IR injury.

Ischemic Preconditioning and Intermittent Ischemia Preserve Bile Flow in a Rat Model of Ischemia Reperfusion Injury

Ischemia and reperfusion (IR) injury of the liver is associated with impaired bile secretion, but the effects of ischemic preconditioning (IPC) and intermittent ischemia (INT) on bile flow are unknown. A rat model of segmental (60%-70%) hepatic ischemia and reperfusion was employed to test the effects of IPC and INT on bile flow. Continuous clamping for 45 min (CC) substantially reduced bile flow, and this did not recover after 60 min of reperfusion. IPC and INT caused a significant recovery of bile flow. The elevation in plasma liver marker enzymes induced by CC was not reduced by IPC and INT. Light microscopy showed mild hepatocyte damage in all groups. In the CC group, the amount of F-actin localized around the bile canaliculi in the ischemic lobes was less than that in the nonischemic lobes, but this difference was not observed in the IPC and INT groups. It is concluded that IPC and INT substantially alleviate the decrease in bile flow induced by ischemia. Bile flow may be useful in the assessment of IR injury.

Direct evidence for neuronal differentiation of astrocyte-like progenitors in the postnatal hippocampus

N-myc downstream-regulated gene 2 (Ndrg2) is a differentiation- and stress-associated molecule predominantly expressed in astrocytes in the central nervous system (CNS). To study the expression and possible role of Ndrg2 in quiescent and activated astrocytes, mice were administrated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP), a Parkinson disease (PD)-related neurotoxin which causes both neurodegeneration and glial activation. Immunohistological analysis revealed that Ndrg2 was highly expressed in both types of astrocytes, but less so in astrocytes during the early process of activation. Ndrg2 was also expressed in astrocyte-like cells, but not in neurons, in human brains from PD and Cortico-basal degeneration (CBD) patients. In cultured astrocytes, gene silencing of Ndrg2 significantly enhanced the numbers of 5-bromo-2′-deoxy-uridine (BrdU)-incorporated and proliferating cell nuclear antigen (PCNA)-positive cells, and reduced the length of cell processes and the amount of F-actin. In contrast, adenovirus-mediated overexpression of Ndrg2 significantly reduced the numbers of BrdU-incorporated and PCNA-positive cells, and enhanced the amount of F-actin. Fractionation and immunocytochemical analysis further revealed that Ndrg2 was located in different cellular fractions including the cytosol and cell surface membranes. These results suggest that Ndrg2 may regulate astroglial activation through the suppression of cell proliferation and stabilization of cell morphology.

Effects of heat stress on Young's modulus of outer hair cells in mice

Intense sound exposure causes permanent hearing loss due to hair cell and cochlear damage. Prior conditioning with sublethal stressors, such as nontraumatic sound, heat stress and restraint protects the ear from acoustic injury. However, the mechanisms underlying conditioning-related cochlear protection remain unknown. In this paper, Young's modulus and the amount of filamentous actin (F-actin) of outer hair cells (OHCs) with/without heat stress were investigated by atomic force microscopy and confocal laser scanning microscopy, respectively. Conditioning with heat stress resulted in a statistically significant increase in Young's modulus of OHCs at 3–6 h after application, and such modulus then began to decrease by 12 h and returned to pre-conditioning level at 48 h after heat stress. The amount of F-actin began to increase by 3 h after heat stress and peaked at 12 h. It then began to decrease by 24 h and returned to the pre-conditioning level by 48–96 h after heat stress. These time courses are consistent with a previous report in which heat stress was shown to suppress permanent threshold shift (PTS). In addition, distortion product otoacoustic emissions (DPOAEs) were confirmed to be enhanced by heat stress. These results suggest that conditioning with heat stress structurally modifies OHCs so that they become stiffer due to an increase in the amount of F-actin. As a consequence, OHCs possibly experience less strain when they are exposed to loud noise, resulting in protection of mammalian hearing from traumatic noise exposure.

Biochemical and Biophysical Studies on the Precursor Cells of Mouse Erythrocytes at Different Stages

The aim of this research is to study the biochemical and biophysical properties of the precursor cells of mouse erythrocytes at different stages and the molecular mechanisms of their regulation. We investigated the degree of terminal differentiation of splenic erythroblasts obtained from mice during the acute phase of disease caused by the anemia-inducing FVAstrain of Friend virus. We analyzed the transcription and protein levels of alpha-globin, beta-globin (erythroid special protein) and GATA-1 (a special erythroid transcription factor). We also have examined the Ca2+ concentration, the distribution and amount of F-actin, important cellular components such as nucleic acids, lipids, and proteins, and the adhesion of precursor cells of RBC at different stages to vascular endothelium. Our results indicated that Ca2+ concentration and the distribution and structure of F-actin changed with the development of proerythroblasts, and that the adhesion rate between the precursor cells and endothelial cells can be correlated with the expression levels of ICAM-1 and P-selectin. These alternations caused changes in biophysical properties of the cell, such as membrane fluidity and deformability.

Rapid Molecular and Morphological Responses of Prostate Cell Lines to Cell–Cell Contact

Cell-cell adhesion in 2-D PZ-HPV-7 prostate epithelial and DU-145 prostate cancer cell aggregates (monolayers), synchronously and rapidly (within 30 s) formed in suspension in an ultrasound trap has been examined over 60 min. The intracellular distributions of the cadherin/catenin complex components for both cell lines were time-dependent and were clearly identifiable as early as 150 s following cell-cell contact in the trap, while equilibrium positions were reached within 60 min following cell-cell contact. The accumulation of E-cadherin at the cell-cell interface was greater for PZ-HPV-7 than for DU-145 cells over 60 min in the trap, with the apparent formation of adherens junctions over that time scale in PZ-HPV-7 but not in DU-145 cells. The amounts of F-actin, alpha-, beta-, and gamma-catenins recruited to the cell-cell interface of PZ-HPV-7 cells were on average 2.4 times higher than those of DU-145 cells. The ability of different cell types to spread along neighboring cells was 1.5-fold greater for the PZ-HPV-7 than for the DU-145 cells. These results, discussed also in the context of earlier studies of cell adhesion in an ultrasound trap, characterize a reduced adhesiveness of DU-145 cells compared to PZ-HPV-7 cells.

Cortactin is essential for F-actin assembly in enteropathogenic Escherichia coli (EPEC)- and enterohaemorrhagic E. coli (EHEC)-induced pedestals and the alpha-helical region is involved in the localization of cortactin to bacterial attachment sites

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are important human pathogens. Upon attachment to host cells, EPEC and EHEC are able to induce actin polymerization, which accumulates, forming a pedestal-like structure beneath the attached bacteria. Using siRNA, we show here that EPEC- and EHEC-induced pedestals are dependent on cortactin, an F-actin-binding protein found in the mammalian cell cortex. Knock-down of cortactin by siRNA resulted in a dramatic reduction of the pedestal formation induced by both pathogens. We also show that disruption of the Src homology 3 (SH3) domain of cortactin, or its downregulation by specific point mutations, negatively affects pedestal formation, suggesting that this domain is important for regulation of F-actin assembly by EPEC and EHEC. Green fluorescent protein (GFP) fused with the SH3 domain (GFP-SH3), proline-rich region (GFP-PRR) or alpha-helical region of cortactin markedly reduced the amount of F-actin at the bacterial attachment sites. Interestingly, neither GFP-SH3 nor GFP-PRR was recruited to the vicinity of the bacterial adherence sites; however, GFP fused to the alpha-helical region was efficiently recruited and colocalized with the attached bacteria. These results demonstrate that cortactin is a requirement for pedestal formation and suggest a novel function for the predicted alpha-helical region of cortactin in actin assembly induced by EPEC and EHEC.

Cdk5 phosphorylates and stabilizes p27kip1 contributing to actin organization and cortical neuronal migration

p27(kip1), a cyclin-dependent kinase (CDK) inhibitor (CKI), generally suppresses CDK activity in proliferating cells. Although another role of p27 in cell migration has been recently suggested in vitro, the physiological importance of p27 in cell migration remains elusive, as p27-deficient mice have not shown any obvious migration-defect-related phenotypes. Here, we show that Cdk5, an unconventional neuronal CDK, phosphorylates and stabilizes p27 as an upstream regulator, maintaining the amount of p27 in post-mitotic neurons. In vivo RNA interference (RNAi) experiments showed that reduced amounts of p27 caused inhibition of cortical neuronal migration and decreased the amount of F-actin in the processes of migrating neurons. The Cdk5-p27 pathway activates an actin-binding protein, cofilin, which is also shown to be involved in cortical neuronal migration in vivo. Our findings shed light on a previously unknown new relationship between CDK and CKI in G0-arrested cells that regulates cytoskeletal reorganization and neuronal migration during corticogenesis.

The C-Terminus of Alpha Spectrin Binds Protein 4.2 and Is Necessary for Optimal Spectrin-Actin Binding.

The red blood cell (RBC) membrane skeleton is composed principally of short F-actin filaments crosslinked by α2β2-spectrin tetramers with the assistance of protein 4.1R. Actin and 4.1R bind to the actin-binding domain (βABD) at the N-terminus of the spectrin β-chain. The adjacent, C-terminal end of α-spectrin, contains a calmodulin-like domain (αCML, aa 2262–2418) that is also called the EF hand domain and is thought to be inert or vestigial. However, the sph1J/sph1J mouse, which has severe hereditary spherocytosis and unstable RBC membranes, makes a mutant α-spectrin that lacks the last 13 amino acids (αCMLΔC13), showing that the domain has some important function. To investigate this function we “fished” for interacting proteins using glutathione-S-transferase (GST)-fused to the CML domain—either the wildtype (αGST-CML) or sph1J (αGST-CMLΔC13). αGST-CML retrieved protein 4.2 from a 2M Tris HCl extract of spectrin-actin depleted human RBC membranes. Protein 4.2 bound αGST-CML with high affinity (Kd = 2.7 x 10−7M) but did not bind αGST-CMLΔC13. Binding was abolished by 1 mM Ca2+, which converts the CML domain to the liganded conformation. The binding site on protein 4.2 localized, at least partly, to amino acids 411–492. Because red cells lacking protein 4.2 are not as severely affected as sph1J/sph1J RBCs, we also tested the effect of the αCMLΔC13 mutation on spectrin-actin binding. A minispectrin was prepared containing the actin-binding domain plus the first four spectrin repeats of the β-chain, combined with the CML domain (±ΔC13) and the last four repeats of the α-chain. The normal and mutant minispectrins were incubated with protein 4.1R, F-actin, or both proteins. The results were striking. The minispectrin containing the normal CML domain bound actin in the presence of protein 4.1R, but the minispectrin containing the mutant CML domain did not. Similarly, the mutant minispectrin was defective in its ability to bind 125I-4.1R in the presence of a constant amount of F-actin. However, the mutation did not affect binding of the minispectrin to protein 4.1R in the absence of actin. We have not yet tested whether protein 4.2 or Ca2+ modulate the effects of the CML domain on spectrin-actin binding. In summary, these experiments clearly show that the calmodulin-like (EF hand) domain of α-spectrin, which was previously considered inert, binds protein 4.2 and also contributes to spectrin-actin binding in the presence of protein 4.1R. Further experiments will be needed to determine whether the CML domain binds actin directly or strengthens the binding of the adjacent actin-binding domain.