Local Actin Filament Geometry Dictates How Myosin Va Molecular Motor Teams Transport Liposomes Through 3D Actin Networks in Vitro

The cell’s dense 3D actin filament network presents numerous challenges to vesicular transport by teams of myosin Va (MyoVa) molecular motors. These teams must navigate their cargo through diverse actin structures ranging from Arp2/3-branched lamellipodial networks to the dense, unbranched cortical networks. To define how actin filament network organization affects MyoVa cargo transport, we created two different 3D actin networks in vitro. One network was comprised of randomly oriented, unbranched actin filaments; the other was comprised of Arp2/3-branched actin filaments, which effectively polarized the network by aligning the actin filament plus-ends. Within both networks, we defined each actin filament’s 3D spatial position using superresolution stochastic optical reconstruction microscopy (STORM) and its polarity by observing the movement of single fluorescent reporter MyoVa. We then characterized the 3D trajectories of fluorescent, 350-nm fluid-like liposomes transported by MyoVa teams (∼10 motors) moving within each of the two networks. Compared with the unbranched network, we observed more liposomes with directed and fewer with stationary motion on the Arp2/3-branched network. This suggests that the modes of liposome transport by MyoVa motors are influenced by changes in the local actin filament polarity alignment within the network. This mechanism was supported by an in silico 3D model that provides a broader platform to understand how cellular regulation of the actin cytoskeletal architecture may fine tune MyoVa-based intracellular cargo transport.

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MYOSIN VA TRANSPORT OF LIPOSOMES IN THREE-DIMENSIONAL ACTIN NETWORKS IS MODULATED BY ACTIN FILAMENT DENSITY, POSITION, AND POLARITY

10.1101/526640 ◽

2019 ◽

Author(s):

Andrew T. Lombardo ◽

Shane R. Nelson ◽

Guy G. Kennedy ◽

Kathleen M. Trybus ◽

Sam Walcott ◽

...

Keyword(s):

Actin Filament ◽

Molecular Motors ◽

Actin Filaments ◽

Three Dimensional ◽

Fluorescent Reporter ◽

Actin Networks ◽

Cargo Transport ◽

Myosin Va ◽

Filament Network

The cell's dense three-dimensional (3D) actin filament network presents numerous challenges to vesicular transport by teams of myosin Va (MyoVa) molecular motors. These teams must navigate their cargo through diverse actin structures ranging from Arp2/3-branched lamellipodial networks to the dense, unbranched cortical networks. To define how actin filament network organization affects MyoVa cargo transport, we created two different 3D actin networks in vitro. One network was comprised of randomly oriented, unbranched actin filaments; the other was comprised of Arp2/3-branched actin filaments, which effectively polarized the network by aligning the actin filament plus-ends. Within both networks, we defined each actin filament's 3D spatial position, using STORM microscopy, and its polarity by observing the movement of single fluorescent, reporter MyoVa. We then characterized the 3D trajectories of fluorescent, 350 nm fluid-like, liposomes transported by MyoVa teams (~10 motors) moving within each of the two networks. Compared to the unbranched network, we observed more liposomes with directed and fewer with stationary motion on the Arp2/3-branched network. This suggests that the modes of liposome transport by MyoVa motors are influenced by changes in the local actin filament polarity alignment within the network. This mechanism was supported by an in silico 3D model that provides a broader platform to understand how cellular regulation of the actin cytoskeletal architecture may fine-tune MyoVa-based intracellular cargo transport.

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Myosin Va Vesicular Transport is Modulated by Actin Filament Density, Orientation, and Polarity in an In Vitro 3D Actin Network

Biophysical Journal ◽

10.1016/j.bpj.2017.11.1182 ◽

2018 ◽

Vol 114 (3) ◽

pp. 211a

Author(s):

Andrew T. Lombardo ◽

Shane R. Nelson ◽

Guy G. Kennedy ◽

Kathleen M. Trybus ◽

Sam Walcott ◽

...

Keyword(s):

Actin Filament ◽

Vesicular Transport ◽

Actin Network ◽

Myosin Va

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MICAL2 acts through Arp3B isoform-specific Arp2/3 complexes to destabilize branched actin networks

10.1101/2020.09.21.306522 ◽

2020 ◽

Author(s):

Chiara Galloni ◽

Davide Carra ◽

Jasmine V. G. Abella ◽

Svend Kjær ◽

Pavithra Singaravelu ◽

...

Keyword(s):

Functional Properties ◽

Actin Filament ◽

Actin Assembly ◽

Actin Network ◽

Network Stability ◽

Actin Networks ◽

Cellular Processes ◽

Actin Turnover ◽

Filament Networks

AbstractThe Arp2/3 complex (Arp2, Arp3 and ARPC1-5) is essential to generate branched actin filament networks for many cellular processes. Human Arp3, ARPC1 and ARPC5 exist as two isoforms but the functional properties of Arp2/3 iso-complexes is largely unexplored. Here we show that Arp3B, but not Arp3 is subject to regulation by the methionine monooxygenase MICAL2, which is recruited to branched actin networks by coronin-1C. Although Arp3 and Arp3B iso-complexes promote actin assembly equally efficiently in vitro, they have different cellular properties. Arp3B turns over significantly faster than Arp3 within the network and upon its depletion actin turnover decreases. Substitution of Arp3B Met293 by Thr, the corresponding residue in Arp3 increases actin network stability, and conversely, replacing Arp3 Thr293 with Gln to mimic Met oxidation promotes network disassembly. Thus, MICAL2 regulates a subset of Arp2/3 complexes to control branched actin network disassembly.

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Myosin 1b Flattens and Prunes Branched Actin Filaments

10.1101/2020.02.26.966663 ◽

2020 ◽

Author(s):

Julien Pernier ◽

Antoine Morchain ◽

Valentina Caorsi ◽

Aurélie Bertin ◽

Hugo Bousquet ◽

...

Keyword(s):

Total Internal Reflection ◽

Molecular Motor ◽

Internal Reflection ◽

Total Internal Reflection Fluorescence ◽

Actin Network ◽

Tirf Microscopy ◽

Actin Networks ◽

Cellular Processes

AbstractMotile and morphological cellular processes require a spatially and temporally coordinated branched actin network that is controlled by the activity of various regulatory proteins including the Arp2/3 complex, profilin, cofilin and tropomyosin. We have previously reported that myosin 1b regulates the density of the actin network in the growth cone. Using in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we show in this report that this molecular motor flattens the Arp2/3-dependent actin branches up to breaking them and reduces the probability to form new branches. This experiment reveals that myosin 1b can produce force sufficient enough to break up the Arp2/3-mediated actin junction. Together with the former in vivo studies, this work emphasizes the essential role played by myosins in the architecture and in the dynamics of actin networks in different cellular regions.Short summaryUsing in vitro F-actin gliding assays and total internal reflection fluorescence (TIRF) microscopy we show that myosin flattens the Arp2/3-dependent actin branches up to breaking them and reduces the probability to form new branches

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The Arp1/11 minifilament of dynactin primes the endosomal Arp2/3 complex

Science Advances ◽

10.1126/sciadv.abd5956 ◽

2021 ◽

Vol 7 (3) ◽

pp. eabd5956 ◽

Cited By ~ 1

Author(s):

Artem I. Fokin ◽

Violaine David ◽

Ksenia Oguievetskaia ◽

Emmanuel Derivery ◽

Caroline E. Stone ◽

...

Keyword(s):

Actin Filament ◽

Actin Networks ◽

Capping Protein ◽

Related Proteins ◽

In Cells

Dendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the capping protein for unclear reasons. Here, we show that the WASH complex interacts with dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins.

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Synergy between Cyclase-associated protein and Cofilin accelerates actin filament depolymerization by two orders of magnitude

Nature Communications ◽

10.1038/s41467-019-13268-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 15

Author(s):

Shashank Shekhar ◽

Johnson Chung ◽

Jane Kondev ◽

Jeff Gelles ◽

Bruce L. Goode

Keyword(s):

Actin Filament ◽

Single Molecule ◽

Actin Filaments ◽

Binding Protein ◽

Actin Dynamics ◽

Actin Network ◽

Cellular Mechanisms ◽

Actin Networks ◽

Binding Event

AbstractCellular actin networks can be rapidly disassembled and remodeled in a few seconds, yet in vitro actin filaments depolymerize slowly over minutes. The cellular mechanisms enabling actin to depolymerize this fast have so far remained obscure. Using microfluidics-assisted TIRF, we show that Cyclase-associated protein (CAP) and Cofilin synergize to processively depolymerize actin filament pointed ends at a rate 330-fold faster than spontaneous depolymerization. Single molecule imaging further reveals that hexameric CAP molecules interact with the pointed ends of Cofilin-decorated filaments for several seconds at a time, removing approximately 100 actin subunits per binding event. These findings establish a paradigm, in which a filament end-binding protein and a side-binding protein work in concert to control actin dynamics, and help explain how rapid actin network depolymerization is achieved in cells.

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Activation of the Arp2/3 Complex by the Actin Filament Binding Protein Abp1p

The Journal of Cell Biology ◽

10.1083/jcb.153.3.627 ◽

2001 ◽

Vol 153 (3) ◽

pp. 627-634 ◽

Cited By ~ 139

Author(s):

Bruce L. Goode ◽

Avital A. Rodal ◽

Georjana Barnes ◽

David G. Drubin

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Mammalian Cells ◽

Actin Assembly ◽

Related Protein ◽

Genetic Studies ◽

Actin Networks ◽

Specific Step

The actin-related protein (Arp) 2/3 complex plays a central role in assembly of actin networks. Because distinct actin-based structures mediate diverse processes, many proteins are likely to make spatially and temporally regulated interactions with the Arp2/3 complex. We have isolated a new activator, Abp1p, which associates tightly with the yeast Arp2/3 complex. Abp1p contains two acidic sequences (DDW) similar to those found in SCAR/WASp proteins. We demonstrate that mutation of these sequences abolishes Arp2/3 complex activation in vitro. Genetic studies indicate that this activity is important for Abp1p functions in vivo. In contrast to SCAR/WASp proteins, Abp1p binds specifically to actin filaments, not monomers. Actin filament binding is mediated by the ADF/cofilin homology (ADF-H) domain of Abp1p and is required for Arp2/3 complex activation in vitro. We demonstrate that Abp1p recruits Arp2/3 complex to the sides of filaments, suggesting a novel mechanism of activation. Studies in yeast and mammalian cells indicate that Abp1p is involved functionally in endocytosis. Based on these results, we speculate that Abp1p may link Arp2/3-mediated actin assembly to a specific step in endocytosis.

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Akt1-associated actomyosin remodelling is required for nuclear lamina dispersal and nuclear shrinkage in epidermal terminal differentiation

Cell Death and Differentiation ◽

10.1038/s41418-020-00712-9 ◽

2021 ◽

Author(s):

Clare Rogerson ◽

Duncan J. Wotherspoon ◽

Cristina Tommasi ◽

Robert W. Button ◽

Ryan F. L. O’Shaughnessy

Keyword(s):

Actin Filament ◽

Molecular Mechanisms ◽

Molecular Motor ◽

Nuclear Lamina ◽

Nuclear Volume ◽

Volume Reduction ◽

Epidermal Keratinocytes ◽

Lamin A ◽

Myosin Iia

AbstractKeratinocyte cornification and epidermal barrier formation are tightly controlled processes, which require complete degradation of intracellular organelles, including removal of keratinocyte nuclei. Keratinocyte nuclear destruction requires Akt1-dependent phosphorylation and degradation of the nuclear lamina protein, Lamin A/C, essential for nuclear integrity. However, the molecular mechanisms that result in complete nuclear removal and their regulation are not well defined. Post-confluent cultures of rat epidermal keratinocytes (REKs) undergo spontaneous and complete differentiation, allowing visualisation and perturbation of the differentiation process in vitro. We demonstrate that there is dispersal of phosphorylated Lamin A/C to structures throughout the cytoplasm in differentiating keratinocytes. We show that the dispersal of phosphorylated Lamin A/C is Akt1-dependent and these structures are specific for the removal of Lamin A/C from the nuclear lamina; nuclear contents and Lamin B were not present in these structures. Immunoprecipitation identified a group of functionally related Akt1 target proteins involved in Lamin A/C dispersal, including actin, which forms cytoskeletal microfilaments, Arp3, required for actin filament nucleation, and Myh9, a component of myosin IIa, a molecular motor that can translocate along actin filaments. Disruption of actin filament polymerisation, nucleation or myosin IIa activity prevented formation and dispersal of cytoplasmic Lamin A/C structures. Live imaging of keratinocytes expressing fluorescently tagged nuclear proteins showed a nuclear volume reduction step taking less than 40 min precedes final nuclear destruction. Preventing Akt1-dependent Lamin A/C phosphorylation and disrupting cytoskeletal Akt1-associated proteins prevented nuclear volume reduction. We propose keratinocyte nuclear destruction and differentiation requires myosin II activity and the actin cytoskeleton for two intermediate processes: Lamin A/C dispersal and rapid nuclear volume reduction.

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Myosin Va interacts with the exosomal protein spermine synthase

Bioscience Reports ◽

10.1042/bsr20182189 ◽

2019 ◽

Vol 39 (3) ◽

Cited By ~ 1

Author(s):

Luciano G. Dolce ◽

Rui M. P. Silva-Junior ◽

Leandro H. P. Assis ◽

Andrey F. Z. Nascimento ◽

Jackeline S. Araujo ◽

...

Keyword(s):

Molecular Motor ◽

Direct Interaction ◽

Tail Domain ◽

Spermine Synthase ◽

Cytoplasmic Vesicles ◽

Myosin Va ◽

Cold Shock Domain ◽

Protein Components ◽

Hybrid Screening

AbstractMyosin Va (MyoVa) is an actin-based molecular motor that plays key roles in the final stages of secretory pathways, including neurotransmitter release. Several studies have addressed how MyoVa coordinates the trafficking of secretory vesicles, but why this molecular motor is found in exosomes is still unclear. In this work, using a yeast two-hybrid screening system, we identified the direct interaction between the globular tail domain (GTD) of MyoVa and four protein components of exosomes: the WD repeat-containing protein 48 (WDR48), the cold shock domain-containing protein E1 (CSDE1), the tandem C2 domain-containing protein 1 (TC2N), and the enzyme spermine synthase (SMS). The interaction between the GTD of MyoVa and SMS was further validated in vitro and displayed a Kd in the low micromolar range (3.5 ± 0.5 µM). SMS localized together with MyoVa in cytoplasmic vesicles of breast cancer MCF-7 and neuroblastoma SH-SY5Y cell lines, known to produce exosomes. Moreover, MYO5A knockdown decreased the expression of SMS gene and rendered the distribution of SMS protein diffuse, supporting a role for MyoVa in SMS expression and targeting.

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