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

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.

Download Full-text

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.

Download Full-text

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

Biophysical Journal ◽

10.1016/j.bpj.2018.11.697 ◽

2019 ◽

Vol 116 (3) ◽

pp. 125a

Author(s):

Sam Walcott ◽

Andrew T. Lombardo ◽

Kathleen M. Trybus ◽

David M. Warshaw

Keyword(s):

Actin Filament ◽

Molecular Motor ◽

Actin Networks ◽

Myosin Va

Download Full-text

Modeling myosin Va liposome transport through actin filament networks reveals a percolation threshold that modulates transport properties

10.1101/2021.08.10.455842 ◽

2021 ◽

Author(s):

Sam Walcott ◽

David M Warshaw

Keyword(s):

Phase Transition ◽

Actin Filament ◽

Actin Filaments ◽

Three Dimensional ◽

Fold Increase ◽

Coarse Grained ◽

Actin Network ◽

Cargo Delivery ◽

Myosin Va ◽

Filament Networks

Myosin Va (myoVa) motors transport membrane-bound cargo through three-dimensional, intracellular actin filament networks. We developed a coarse-grained, in silico model to predict how actin filament density (3-800 filaments) within a randomly oriented actin network affects fluid-like liposome (350nm vs. 1,750nm) transport by myoVa motors. 5,000 simulated liposomes transported within each network adopted one of three states: transport, tug of war, or diffusion. Diffusion due to liposome detachment from actin rarely occurred given at least 10 motors on the liposome surface. However, with increased actin density, liposomes transitioned from primarily directed transport on single actin filaments to an apparent random walk, resulting from a mixture of transport and tug of wars as the probability of encountering additional actin filaments increased. This phase transition arises from a percolation phase transition at a critical number of accessible actin filaments, Nc. Nc, is a geometric property of the actin network that depends only on the position and polarity of the actin filaments and the liposome diameter, as evidenced by a five-fold increase in liposome diameter resulting in a five-fold decrease in Nc. Thus, in cells, actin network density and cargo size may be regulated to match cargo delivery to the cell's physiological demands.

Download Full-text

Modeling myosin Va liposome transport through actin filament networks reveals a percolation threshold that modulates transport properties

Molecular Biology of the Cell ◽

10.1091/mbc.e21-08-0389 ◽

2021 ◽

Author(s):

S. Walcott ◽

D. M. Warshaw

Keyword(s):

Phase Transition ◽

Actin Filament ◽

Actin Filaments ◽

Three Dimensional ◽

Fold Increase ◽

Coarse Grained ◽

Actin Network ◽

Transport Distance ◽

Myosin Va ◽

Filament Networks

Myosin Va (myoVa) motors transport membrane-bound cargo through three-dimensional, intracellular actin filament networks. We developed a coarse-grained, in silico model to predict how actin filament density (3-800 filaments) within a randomly oriented actin network affects fluid-like liposome (350nm vs. 1,750nm) transport by myoVa motors. 5,000 simulated liposomes transported within each network adopted one of three states: transport, tug of war, or diffusion. Diffusion due to liposome detachment from actin rarely occurred given at least 10 motors on the liposome surface. However, with increased actin density, liposomes transitioned from primarily directed transport on single actin filaments to an apparent random walk, resulting from a mixture of transport and tug of wars as the probability of encountering additional actin filaments increased. This phase transition arises from a percolation phase transition at a critical number of accessible actin filaments, Nc. Nc is a geometric property of the actin network that depends only on the position and polarity of the actin filaments, transport distance, and the liposome diameter, as evidenced by a five-fold increase in liposome diameter resulting in a five-fold decrease in Nc. Thus, in cells, actin network density and cargo size may be regulated to match cargo delivery to the cell's physiological demands. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Download Full-text

Myosin Va transport of liposomes in three-dimensional actin networks is modulated by actin filament density, position, and polarity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1901176116 ◽

2019 ◽

Vol 116 (17) ◽

pp. 8326-8335 ◽

Cited By ~ 6

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 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.

Download Full-text

Myosin Va molecular motors manoeuvre liposome cargo through suspended actin filament intersections in vitro

Nature Communications ◽

10.1038/ncomms15692 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 22

Author(s):

Andrew T. Lombardo ◽

Shane R. Nelson ◽

M. Yusuf Ali ◽

Guy G. Kennedy ◽

Kathleen M. Trybus ◽

...

Keyword(s):

Actin Filament ◽

Molecular Motors ◽

Myosin Va

Download Full-text

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.

Download Full-text

Endoplasmic reticulum degradation of a subunit of the asialoglycoprotein receptor in vitro. Vesicular transport from endoplasmic reticulum is unnecessary.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)48445-1 ◽

1992 ◽

Vol 267 (1) ◽

pp. 5-8 ◽

Cited By ~ 5

Author(s):

L WikstrɆm ◽

H F Lodish

Keyword(s):

Endoplasmic Reticulum ◽

Vesicular Transport ◽

Asialoglycoprotein Receptor ◽

A Subunit

Download Full-text

A novel role for WAVE1 in controlling actin network growth rate and architecture

Molecular Biology of the Cell ◽

10.1091/mbc.e14-10-1477 ◽

2015 ◽

Vol 26 (3) ◽

pp. 495-505 ◽

Cited By ~ 9

Author(s):

Meredith O. Sweeney ◽

Agnieszka Collins ◽

Shae B. Padrick ◽

Bruce L. Goode

Keyword(s):

Actin Filament ◽

Specific Activity ◽

Inhibitory Effect ◽

Actin Network ◽

Inhibitory Effects ◽

Cellular Functions ◽

Network Growth ◽

Assembly Kinetics ◽

Filament Networks ◽

First Time

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 (“V”) domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2.

Download Full-text