Interdependence of Filament Nucleation, Elongation and Crosslinking in Actin Bundle Assembly

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.

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A binding protein regulates myosin-7a dimerization and actin bundle assembly

Nature Communications ◽

10.1038/s41467-020-20864-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Rong Liu ◽

Neil Billington ◽

Yi Yang ◽

Charles Bond ◽

Amy Hong ◽

...

Keyword(s):

Atpase Activity ◽

Actin Filament ◽

Single Molecule ◽

Binding Protein ◽

Living Cells ◽

Actin Bundle ◽

Actin Bundles ◽

Cytoskeletal Remodeling ◽

Actin Protrusions

AbstractMyosin-7a, despite being monomeric in isolation, plays roles in organizing actin-based cell protrusions such as filopodia, microvilli and stereocilia, as well as transporting cargoes within them. Here, we identify a binding protein for Drosophila myosin-7a termed M7BP, and describe how M7BP assembles myosin-7a into a motile complex that enables cargo translocation and actin cytoskeletal remodeling. M7BP binds to the autoinhibitory tail of myosin-7a, extending the molecule and activating its ATPase activity. Single-molecule reconstitution show that M7BP enables robust motility by complexing with myosin-7a as 2:2 translocation dimers in an actin-regulated manner. Meanwhile, M7BP tethers actin, enhancing complex’s processivity and driving actin-filament alignment during processive runs. Finally, we show that myosin-7a-M7BP complex assembles actin bundles and filopodia-like protrusions while migrating along them in living cells. Together, these findings provide insights into the mechanisms by which myosin-7a functions in actin protrusions.

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Actin-binding proteins sensitively mediate actin bundle stiffness

Journal of Biomechanics ◽

10.1016/s0021-9290(06)83904-7 ◽

2006 ◽

Vol 39 ◽

pp. S240

Author(s):

M. Bathe ◽

M. Claessens ◽

E. Frey ◽

A. Bausch

Keyword(s):

Binding Proteins ◽

Actin Binding ◽

Actin Binding Proteins ◽

Actin Bundle

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Development of macrociliary cells in Beroe. I. Actin bundles and centriole migration

Journal of Cell Science ◽

10.1242/jcs.89.1.67 ◽

1988 ◽

Vol 89 (1) ◽

pp. 67-80

Author(s):

S. Tamm ◽

S.L. Tamm

Keyword(s):

Cell Membrane ◽

Basal Body ◽

Actin Filaments ◽

Close Association ◽

Directed Assembly ◽

Double Layers ◽

Basal Bodies ◽

Apical Surface ◽

Actin Bundle ◽

Microfilament Bundle

Differentiation of macrociliary cells on regenerating lips of the ctenophore Beroe was studied by transmission electron microscopy. In this study of early development, we found that basal bodies for macrocilia arise by an acentriolar pathway near the nucleus and Golgi apparatus, in close association with plaques of dense fibrogranular bodies. Procentrioles are often aligned side-by-side in double layers with the cartwheel ends facing outward toward the surrounding plaques of dense granules. Newly formed basal bodies then disband from groups and develop a long striated rootlet at one end. At the same time, an array of microfilaments arises in the basal cytoplasm. The microfilaments are arranged in parallel strands oriented toward the cell surface. The basal body-rootlet units are transported to the apical surface in close association with the assembling actin filament bundle. Microfilaments run parallel to and alongside the striated rootlets, to which they often appear attached. Basal body-rootlet units migrate at the heads of trails of microfilaments, as if they are pushed upwards by elongation of their attached actin filaments. Near the apical surface the actin bundle curves and runs below the cell membrane. Newly arrived basal body-rootlets tilt upwards out of the microfilament bundle to contact the cell membrane and initiate ciliogenesis. The basal bodies tilt parallel to the flat sides of the rootlets, and away from the direction in which the basal feet point. The actin bundle continues to enlarge during ciliogenesis. These results suggest that basal body migration may be driven by the directed assembly of attached actin filaments.

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Helical buckling of actin inside filopodia generates traction

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1411761112 ◽

2014 ◽

Vol 112 (1) ◽

pp. 136-141 ◽

Cited By ~ 34

Author(s):

Natascha Leijnse ◽

Lene B. Oddershede ◽

Poul M. Bendix

Keyword(s):

Cell Membrane ◽

Rotational Motion ◽

Actin Filaments ◽

Cell Communication ◽

Confocal Imaging ◽

Actin Bundle ◽

Cellular Processes ◽

Membrane Protrusions ◽

A Cell ◽

Helical Buckling

Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell–cell communication. Filopodia contain cross-linked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle.

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Massive actin bundle couples macrocilia to muscles in the ctenophoreBeroë

Cell Motility and the Cytoskeleton ◽

10.1002/cm.970070204 ◽

1987 ◽

Vol 7 (2) ◽

pp. 116-128 ◽

Cited By ~ 12

Author(s):

Sidney L. Tarmm ◽

Signhild Tamm

Keyword(s):

Actin Bundle

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Bundling of actin filaments by alpha-actinin depends on its molecular length.

The Journal of Cell Biology ◽

10.1083/jcb.110.6.2013 ◽

1990 ◽

Vol 110 (6) ◽

pp. 2013-2024 ◽

Cited By ~ 169

Author(s):

R K Meyer ◽

U Aebi

Keyword(s):

Smooth Muscle ◽

Actin Filament ◽

Actin Filaments ◽

Actin Binding ◽

Molar Ratio ◽

Cross Linking ◽

Actin Bundle ◽

Actin Bundles ◽

Chicken Gizzard ◽

Molecular Length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.

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Coincident microvillar actin bundle disruption and perinuclear actin sequestration in anoxic proximal tubule

AJP Renal Physiology ◽

10.1152/ajprenal.2000.278.6.f886 ◽

2000 ◽

Vol 278 (6) ◽

pp. F886-F893 ◽

Cited By ~ 15

Author(s):

Peter White ◽

R. Brian Doctor ◽

Rolf H. Dahl ◽

Jing Chen

Keyword(s):

Control Level ◽

Dependent Manner ◽

Differential Interference Contrast ◽

Differential Interference Contrast Microscopy ◽

Proximal Tubular Cells ◽

Actin Bundle ◽

Tubular Cells ◽

Contrast Microscopy ◽

Proximal Tubular ◽

Interference Contrast Microscopy

The present studies investigated acute disruption of microvillar actin cytoskeleton and actin association with other cytoskeletal components in ATP-depleted rabbit proximal tubular cells. Video-enhanced differential-interference contrast microscopy and confocal microscopy were used to follow the fate of F-actin during the disruption of microvilli. Within individual cells, all microvilli collapsed simultaneously. Microvillar actin filaments underwent a parallel decrease in length. Using a sequential cytoskeletal extraction protocol and electron microscopy, we revealed in the present studies the coincident sequestration of a distinct, perinuclear pool of actin that was primarily absent in control cells. Actin sequestration progressed in a duration-dependent manner, occurring as early as 15 min of anoxia when cellular ATP dropped to <5% of control level. Phalloidin staining and depolymerization treatment showed the majority (>90%) of this sequestered actin to be F-actin. A microvillar actin bundling protein villin was also sequestered in the same perinuclear complex of anoxic proximal tubules. In conclusion, the present results demonstrate a coincident microvillar actin bundle disruption and the perinuclear sequestration of F-actin in ATP-depleted proximal tubular cells.

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F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments.

The Journal of Cell Biology ◽

10.1083/jcb.135.5.1291 ◽

1996 ◽

Vol 135 (5) ◽

pp. 1291-1308 ◽

Cited By ~ 55

Author(s):

L G Tilney ◽

P Connelly ◽

S Smith ◽

G M Guild

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Modular Design ◽

Thin Sections ◽

Actin Bundle ◽

Actin Bundles ◽

Phalloidin Staining ◽

Filament Bundles ◽

End To End ◽

As If

The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper.

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Biomechanical Aspects of Actin Bundle Dynamics

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.00422 ◽

2020 ◽

Vol 8 ◽

Author(s):

Julia Lange ◽

Erik Bernitt ◽

Hans-Günther Döbereiner

Keyword(s):

Actin Bundle

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