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Prominent actin fiber arrays in Drosophila tendon cells represent architectural elements different from stress fibers.

ABSTRACT: Tendon cells are specialized cells of the insect epidermis that connect basally attached muscle tips to the cuticle on their apical surface via prominent arrays of microtubules. Tendon cells of Drosophila have become a useful genetic model system to address questions with relevance to cell and developmental biology. Here, we use light, confocal, and electron microscopy to present a refined model of the subcellular organization of tendon cells. We show that prominent arrays of F-actin exist in tendon cells that fully overlap with the microtubule arrays, and that type II myosin accumulates in the same area. The F-actin arrays in tendon cells seem to represent a new kind of actin structure, clearly distinct from stress fibers. They are highly resistant to F-actin-destabilizing drugs, to the application of myosin blockers, and to loss of integrin, Rho1, or mechanical force. They seem to represent an important architectural element of tendon cells, because they maintain a connection between apical and basal surfaces even when microtubule arrays of tendon cells are dysfunctional. Features reported here and elsewhere for tendon cells are reminiscent of the structural and molecular features of support cells in the inner ear of vertebrates, and they might have potential translational value.

SUBMITTER: Alves-Silva J

PROVIDER: S-EPMC2555930 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Calcium-calmodulin-dependent kinase II (CaMKII) has an important role in dendritic spine remodeling upon synaptic stimulation. Using fluorescence video microscopy and image analysis, we investigated the architectural dynamics of rhodamine-phalloidin stabilized filamentous actin (F-actin) networks cross-linked by CaMKII. We used automated image analysis to identify F-actin bundles and crossover junctions and developed a dimensionless metric to characterize network architecture. Similar networks were formed by three different CaMKII species with a 10-fold length difference in the linker region between the kinase domain and holoenzyme hub, implying linker length is not a primary determinant of F-actin cross-linking. Electron micrographs showed that at physiological molar ratios, single CaMKII holoenzymes cross-linked multiple F-actin filaments at random, whereas at higher CaMKII/F-actin ratios, filaments bundled. Light microscopy established that the random network architecture resisted macromolecular crowding with polyethylene glycol and blocked ATP-powered compaction by myosin-II miniature filaments. Importantly, the networks disassembled after the addition of calcium-calmodulin and were then spaced within 3 min into compacted foci by myosin motors or more slowly (30 min) aggregated by crowding. Single-molecule total internal reflection fluorescence microscopy showed CaMKII dissociation from surface-immobilized globular actin exhibited a monoexponential dwell-time distribution, whereas CaMKII bound to F-actin networks had a long-lived fraction, trapped at crossover junctions. Release of CaMKII from F-actin, triggered by calcium-calmodulin, was too rapid to measure with flow-cell exchange (<20 s). The residual bound fraction was reduced substantially upon addition of an N-methyl-D-aspartate receptor peptide analog but not ATP. These results provide mechanistic insights to CaMKII-actin interactions at the collective network and single-molecule level. Our findings argue that CaMKII-actin networks in dendritic spines maintain spine size against physical stress. Upon synaptic stimulation, CaMKII is disengaged by calcium-calmodulin, triggering network disassembly, expansion, and subsequent compaction by myosin motors with kinetics compatible with the times recorded for the poststimulus changes in spine volume.

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Prominent actin fiber arrays in Drosophila tendon cells represent architectural elements different from stress fibers.

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