scholarly journals Simultaneous quantification of actin monomer and filament dynamics with modeling-assisted analysis of photoactivation

2016 ◽  
Vol 129 (24) ◽  
pp. 4633-4643 ◽  
Author(s):  
Maryna Kapustina ◽  
Tracy-Ann Read ◽  
Eric A. Vitriol
Keyword(s):  
Actin Monomer ◽  
Simultaneous Quantification ◽  
Filament Dynamics ◽  
Assisted Analysis

scholarly journals The C-terminal dimerization motif of cyclase-associated protein is essential for actin monomer regulation

2016 ◽  
Vol 473 (23) ◽  
pp. 4427-4441 ◽  
Author(s):  
Shohei Iwase ◽  
Shoichiro Ono
Keyword(s):  
Regulatory Protein ◽  
Full Length ◽  
Regulatory Function ◽  
Actin Monomer ◽  
Nucleotide Exchange ◽  
Strong Activity ◽  
Filament Dynamics ◽  
Evolutionarily Conserved ◽  
Syndrome Protein

Cyclase-associated protein (CAP) is a conserved actin-regulatory protein that functions together with actin depolymerizing factor (ADF)/cofilin to enhance actin filament dynamics. CAP has multiple functional domains, and the function to regulate actin monomers is carried out by its C-terminal half containing a Wiskott–Aldrich Syndrome protein homology 2 (WH2) domain, a CAP and X-linked retinitis pigmentosa 2 (CARP) domain, and a dimerization motif. WH2 and CARP are implicated in binding to actin monomers and important for enhancing filament turnover. However, the role of the dimerization motif is unknown. Here, we investigated the function of the dimerization motif of CAS-2, a CAP isoform in the nematode Caenorhabditis elegans, in actin monomer regulation. CAS-2 promotes ATP-dependent recycling of ADF/cofilin-bound actin monomers for polymerization by enhancing exchange of actin-bound nucleotides. The C-terminal half of CAS-2 (CAS-2C) has nearly as strong activity as full-length CAS-2. Maltose-binding protein (MBP)-tagged CAS-2C is a dimer. However, MBP-CAS-2C with a truncation of either one or two C-terminal β-strands is monomeric. Truncations of the dimerization motif in MBP-CAS-2C nearly completely abolish its activity to sequester actin monomers from polymerization and enhance nucleotide exchange on actin monomers. As a result, these CAS-2C variants, also in the context of full-length CAS-2, fail to compete with ADF/cofilin to release actin monomers for polymerization. CAS-2C variants lacking the dimerization motif exhibit enhanced binding to actin filaments, which is mediated by WH2. Taken together, these results suggest that the evolutionarily conserved dimerization motif of CAP is essential for its C-terminal region to exert the actin monomer-specific regulatory function.

scholarly journals ATP-dependent regulation of actin monomer–filament equilibrium by cyclase-associated protein and ADF/cofilin

2013 ◽  
Vol 453 (2) ◽  
pp. 249-259 ◽  
Author(s):  
Kazumi Nomura ◽  
Shoichiro Ono
Keyword(s):  
Actin Filaments ◽  
Striated Muscle ◽  
Actin Binding ◽  
Molar Ratio ◽  
Dependent Manner ◽  
Actin Monomer ◽  
Nucleotide Exchange ◽  
C Elegans ◽  
Filament Dynamics ◽  
Steady State Levels

CAP (cyclase-associated protein) is a conserved regulator of actin filament dynamics. In the nematode Caenorhabditis elegans, CAS-1 is an isoform of CAP that is expressed in striated muscle and regulates sarcomeric actin assembly. In the present study, we report that CAS-2, a second CAP isoform in C. elegans, attenuates the actin-monomer-sequestering effect of ADF (actin depolymerizing factor)/cofilin to increase the steady-state levels of actin filaments in an ATP-dependent manner. CAS-2 binds to actin monomers without a strong preference for either ATP– or ADP–actin. CAS-2 strongly enhances the exchange of actin-bound nucleotides even in the presence of UNC-60A, a C. elegans ADF/cofilin that inhibits nucleotide exchange. UNC-60A induces the depolymerization of actin filaments and sequesters actin monomers, whereas CAS-2 reverses the monomer-sequestering effect of UNC-60A in the presence of ATP, but not in the presence of only ADP or the absence of ATP or ADP. A 1:100 molar ratio of CAS-2 to UNC-60A is sufficient to increase actin filaments. CAS-2 has two independent actin-binding sites in its N- and C-terminal halves, and the C-terminal half is necessary and sufficient for the observed activities of the full-length CAS-2. These results suggest that CAS-2 (CAP) and UNC-60A (ADF/cofilin) are important in the ATP-dependent regulation of the actin monomer–filament equilibrium.

scholarly journals Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin

2001 ◽  
Vol 155 (2) ◽  
pp. 251-260 ◽  
Author(s):  
Sandra Palmgren ◽  
Pauli J. Ojala ◽  
Martin A. Wear ◽  
John A. Cooper ◽  
Pekka Lappalainen
Keyword(s):  
Actin Filament ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Actin Monomer ◽  
Cortical Actin ◽  
Gene Encoding ◽  
Capping Protein ◽  
Filament Dynamics ◽  
Filament Assembly

Twinfilin is a ubiquitous actin monomer–binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the α and β subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer–sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

Computer-Assisted Analysis of Multi-Color Confocal Microscopic Datasets: Automated Light Microscopic Discrimination of Synaptic Relationships

1996 ◽  
Vol 54 ◽  
pp. 284-285
Author(s):  
M Wessendorf ◽  
A Beuning ◽  
D Cameron ◽  
J Williams ◽  
C Knox
Keyword(s):  
Nerve Fibers ◽  
Confocal Scanning Laser Microscopy ◽  
Computer Assisted ◽  
Nerve Terminals ◽  
Neuronal Somata ◽  
Scanning Laser Microscopy ◽  
Confocal Scanning ◽  
Entire Cell ◽  
Confocal Scanning Laser ◽  
Assisted Analysis

Multi-color confocal scanning-laser microscopy (CSLM) allows examination of the relationships between neuronal somata and the nerve fibers surrounding them at sub-micron resolution in x,y, and z. Given these properties, it should be possible to use multi-color CSLM to identify relationships that might be synapses and eliminate those that are clearly too distant to be synapses. In previous studies of this type, pairs of images (e.g., red and green images for tissue stained with rhodamine and fluorescein) have been merged and examined for nerve terminals that appose a stained cell (see, for instance, Mason et al.). The above method suffers from two disadvantages, though. First, although it is possible to recognize appositions in which the varicosity abuts the cell in the x or y axes, it is more difficult to recognize them if the apposition is oriented at all in the z-axis—e.g., if the varicosity lies above or below the neuron rather than next to it. Second, using this method to identify potential appositions over an entire cell is time-consuming and tedious.

Structure elucidation by NMR spectroscopy: Assisted analysis from spectra to molecular structure

Planta Medica ◽  
2014 ◽  
Vol 80 (10) ◽  
Author(s):  
S Groscurth ◽  
T Kühn ◽  
P Kessler ◽  
V Rukachaisirikul
Keyword(s):  
Molecular Structure ◽  
Nmr Spectroscopy ◽  
Structure Elucidation ◽  
Assisted Analysis

HS-SPME/GC-MS Assisted Analysis of Volatile Constituents in Different Strains of Shiitake Culinary- Medicinal Mushroom, Lentinus edodes (Agaricomycetes)

2019 ◽  
Vol 21 (7) ◽  
pp. 693-702
Author(s):  
Lívia Martinez Abreu Soares Costa ◽  
Maiara Andrade de Carvalho Sousa ◽  
Thiago Pereira Souza ◽  
Whasley Ferreira Duarte ◽  
Diego Cunha Zied ◽  
...  
Keyword(s):  
Medicinal Mushroom ◽  
Lentinus Edodes ◽  
Volatile Constituents ◽  
Different Strains ◽  
Assisted Analysis
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