Transmembrane Signaling Regulates the Remodeling of the Actin Cytoskeleton:Roles of PKC and Adducin

Members of the conventional protein kinase C (cPKC) family are activated by both DAG and Ca2+ and have been implicated in the regulation of the actin cytoskeleton. Drosophila contains two cPKCs, Pkc53E (Pkc1) and eye-PKC (Pkc2); mutants missing each PKC lead to retinal degeneration. While eye-PKC is critical for the visual signaling, the role of Pkc53E is not known. We identified a photoreceptor-specific isoform of Pkc53E and show Pkc53E-RNAi negatively impacts the actin cytoskeleton of rhabdomeres. Interestingly, Pkc53E-RNAi enhances the degeneration of norpAP24 photoreceptors, suggesting Pkc53E could be activated independently of NorpA/PLCβ4. We further demonstrate that in norpAP24 photoreceptors Plc21C can be activated by Gq, which is responsible for the activation of Pkc53E. We explored whether Pkc53E regulates adducin in Drosophila photoreceptors. Adducin cross-links the actin cytoskeleton to the spectrin network, which is blunted by PKC phosphorylation. Importantly, we observed that phosphorylation of adducin was greatly reduced in a null allele of pkc53E. Downregulation of hts that encodes Drosophila adducin, exerts a more severe effect than Pkc53E-RNAi to impact the actin cytoskeleton. In contrast, overexpression of a mCherry tagged Add2, one of the three Drosophila adducin isoforms, led to the apical expansion of rhabdomeres with overgrowth of the actin cytoskeleton. This phenotype is likely caused by the dominant-negative activity of the tagged Add2 as it also was observed in α-spectrin-RNAi or β-spectrin-RNAi. Interestingly, downregulation of Pkc53E does not suppress the expansion of rhabdomeres during development, but negatively affects the appearance of rhabdomeres in adult photoreceptors. We conclude that Drosophila adducin has two distinct functions: in pupal photoreceptors, it regulates rhabdomere morphogenesis, which is independent of Pkc53E. In adult photoreceptors, it promotes the maintenance of the actin cytoskeleton, which is regulated by Pkc53E in response to the light-induced activation of the PLCβ activity.

Endothelial integration of mechanosensory signals by the spectrin cytoskeleton

Physiological blood flow induces the secretion of vasoactive compounds, notably NO, and promotes endothelial cell elongation and reorientation parallel to the direction of applied shear. How shear is sensed and relayed to intracellular effectors is incompletely understood. We demonstrate that an apical spectrin network is essential to convey the force imposed by shear to endothelial mechanosensors. By anchoring CD44, spectrin modulates the cell surface density of hyaluronan, which senses and translates shear into changes in plasma membrane tension. Spectrins also regulate the stability of apical caveolae, where the mechanosensitive Piezo1 channels are thought to reside. Accordingly, shear-induced Piezo1 activation and the associated calcium influx were absent in spectrin-deficient cells. As a result, cell realignment and flow-induced eNOS stimulation were similarly dependent on spectrin. We concluded that the apical spectrin network is not only required for shear sensing, but transmits and distributes the resulting tensile forces to mechanosensors that elicit protective and vasoactive responses.

Endothelial integration of mechanosensory signals by the spectrin cytoskeleton

Physiological blood flow induces the secretion of vasoactive compounds, notably NO, and promotes endothelial cell elongation and reorientation parallel to the direction of applied shear. How shear is sensed and relayed to intracellular effectors is incompletely understood. We demonstrate that an apical spectrin network is essential to convey the force imposed by shear to endothelial mechanosensors. By anchoring CD44, spectrin modulates the cell surface density of hyaluronan, which senses and translates shear into changes in plasma membrane tension. Spectrins also regulate the stability of apical caveolae, where the mechanosensitive Piezo1 channels are thought to reside. Accordingly, shear-induced Piezo1 activation and the associated calcium influx were absent in spectrin-deficient cells. As a result, cell realignment and flow-induced eNOS stimulation were similarly dependent on spectrin. We concluded that the apical spectrin network is not only required for shear sensing, but transmits and distributes the resulting tensile forces to mechanosensors that elicit protective and vasoactive responses.

Mechanical Stretch Inhibition Sensitizes Proprioceptors to Compressive Stresses

A repetitive gait cycle is an archetypical component within the behavioural repertoire of many if not all animals including humans. It originates from mechanical feedback within proprioceptors to adjust the motorprogram during locomotion and thus leads to a periodic orbit in a low dimensional space. Here, we investigate the mechanics, molecules and neurons responsible for proprioception in Caenorhabditis (C.) elegans to gain insight into how mechanosensation shapes the orbital trajectory to a well-defined limit cycle. We used genome editing, force spectroscopy and multiscale modeling and found that alternating tension and compression with the spectrin network of a single proprioceptor encodes body posture and informs TRP-4/NOMPC and TWK-16/TREK2 homologs of mechanosensitive ion channels during locomotion. In contrast to a widely accepted model of proprioceptive ‘stretch’ reception, we found that proprioceptors activated under compressive stresses in vivo and in vitro, and speculate that this property is conserved across function and species.

A presynaptic spectrin network controls active zone assembly and neurotransmitter release

ABSTRACTWe have previously reported that Drosophila Tenectin (Tnc) recruits αPS2/βPS integrin to ensure structural and functional integrity at larval NMJs (Wang et al., 2018). In muscles, Tnc/integrin engages the spectrin network to regulate the size and architecture of synaptic boutons. In neurons, Tnc/integrin controls neurotransmitter release. Here we show that presynaptic Tnc/integrin modulates the synaptic accumulation of key active zone components, including the Ca2+ channel Cac and the active zone scaffold Brp. Presynaptic α-Spectrin appears to be both required and sufficient for the recruitment of Cac and Brp. We visualized the endogenous α-Spectrin and found that Tnc controls spectrin recruitment at synaptic terminals. Thus, Tnc/integrin anchors the presynaptic spectrin network and ensures the proper assembly and function of the active zones. Since pre- and postsynaptic Tnc/integrin limit each other, we hypothesize that this pathway links dynamic changes within the synaptic cleft to changes in synaptic structure and function.

Conformational Distortions of the Red Blood Cell Spectrin Matrix Nanostructure in Response to Temperature Changes In Vitro

The spectrin matrix is a structural element of red blood cells (RBCs). As such, it affects RBC morphology, membrane deformability, nanostructure, stiffness, and, ultimately, the rheological properties of blood. However, little is known about how temperature affects the spectrin matrix. In this study, the nanostructure of the spectrin network was recorded by atomic force microscopy. We describe how the nanostructure of the RBC spectrin matrix changes from a regular network to a chaotic pattern following an increase in temperature from 20 to 50°C. At 20–37°С, the spectrin network formed a regular structure with dimensions of typically 150±60 nm. At 42–43°С, 83% of the spectrin network assumed an irregular structure. Finally, at 49–50°С the chaotic pattern was observed, and no quantitative estimates of the spectrin structure’s parameters could be made. These results can be useful for biophysical studies on the destruction of the spectrin network under pathological conditions, as well as for investigating cell morphology and blood rheology in different diseases.

The actin-capping protein alpha-adducin is required for T-cell costimulation

Alpha-adducin (Add1) is a critical component of the actin-spectrin network in erythrocytes, acting to cap the fast-growing, barbed ends of actin filaments, and recruiting spectrin to these junctions. Add1 is highly expressed in T cells, but its role in T-cell activation has not been examined. Using a conditional knockout model, we show that Add1 is necessary for complete activation of CD4+ T cells in response to low levels of antigen but is dispensable for CD8+ T cell activation and response to infection. Surprisingly, costimulatory signals through CD28 were completely abrogated in the absence of Add1. This study is the first to examine the role of actin-capping in T cells, and it reveals a previously unappreciated role for the actin cytoskeleton in regulating costimulation.

Structural diversity of erythrocytes in patients with hereditary spherocytosis

Objective: to study the shape of erythrocytes and structure of their surface layer including the membrane and cytoskeleton (actin-spectrin network) in child patients with hereditary spherocytosis. Material and methods. The methods of optic and atomic-force microscopy were used in the study. Results. A variety of erythrocyte shapes with such prevalent types as discocytes, spherocytes, and echinocytes were revealed in the blood of the patients. The surface of certain cells contained microvesicules. The spatial heterogeneity of the structure of mechanical property maps of the cell surface layer was detected. Conclusion. The diversity of erythrocyte features in patients with hereditary spherocytosis is present both at the level of the cell shapes and at the level of the structure of mechanical property maps of their surface layer.

Effect of spectrin network elasticity on the shapes of erythrocyte doublets

Red blood cells (RBCs) in plasma or polymer solution interact attractively to form various shapes of RBC doublets. A rich variety of doublet shapes is found, depending on membrane shear and bending elasticity, reduced volumes, and adhesion strength.