High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

1992 ◽  
Vol 50 (1) ◽  
pp. 512-513
Author(s):  
J. Jakana ◽  
M.F. Schmid ◽  
P. Matsudaira ◽  
W. Chiu
Keyword(s):  
High Resolution ◽  
Binding Proteins ◽  
Actin Binding ◽  
Eukaryotic Cells ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Actin Bundle ◽  
Actin Bundles ◽  
3 Dimensional ◽  
Macromolecular 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.

Actin-binding proteins sensitively mediate actin bundle stiffness

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

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

1990 ◽  
Vol 110 (6) ◽  
pp. 2013-2024 ◽  
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molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium.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.

The high-resolution crystal structure of phosphatidylinositol 4-kinase IIβ and the crystal structure of phosphatidylinositol 4-kinase IIα containing a nucleoside analogue provide a structural basis for isoform-specific inhibitor design

2015 ◽  
Vol 71 (7) ◽  
pp. 1555-1563 ◽  
Author(s):  
Martin Klima ◽  
Adriana Baumlova ◽  
Dominika Chalupska ◽  
Hubert Hřebabecký ◽  
Milan Dejmek ◽  
...  
Keyword(s):  
Crystal Structure ◽  
High Resolution ◽  
Nucleoside Analogue ◽  
Specific Inhibitor ◽  
Eukaryotic Cells ◽  
Structural Basis ◽  
Conformational Heterogeneity ◽  
Inhibitor Design ◽  
Hydrophobic Pocket ◽  
Phosphatidylinositol 4

Phosphatidylinositol 4-phosphate (PI4P) is the most abundant monophosphoinositide in eukaryotic cells. Humans have four phosphatidylinositol 4-kinases (PI4Ks) that synthesize PI4P, among which are PI4K IIβ and PI4K IIα. In this study, two crystal structures are presented: the structure of human PI4K IIβ and the structure of PI4K IIα containing a nucleoside analogue. The former, a complex with ATP, is the first high-resolution (1.9 Å) structure of a PI4K. These structures reveal new details such as high conformational heterogeneity of the lateral hydrophobic pocket of the C-lobe and together provide a structural basis for isoform-specific inhibitor design.

Comparison of 13Å helical and 7Å crystallographic projection maps of frozen, hydrated acrosomal bundle from limulus sperm

1995 ◽  
Vol 53 ◽  
pp. 850-851
Author(s):  
Michael F. Schmid ◽  
Joanita Jakana ◽  
Paul Matsudaira ◽  
Wah Chiu
Keyword(s):  
Extracellular Matrix ◽  
Actin Filament ◽  
Binding Sites ◽  
Repeat Unit ◽  
Nutrient Transport ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Cross Linking ◽  
3 Dimensional ◽  
Placental Protein

Actin associates with crosslinking proteins to form bundles and networks. These assemblies can provide a scaffold to anchor organelles, a support for the cell membrane or can attach the cell to the extracellular matrix or to other cells. Each crosslinker must have at least two actin binding sites, one for each actin filament. Scruin, in the Limulus acrosomal bundle, shows no homology with other actin cross-linking proteins. However, it has two homologous domains also found in the sequence encoded by kelch, a gene in Drosophila that is important in nutrient transport into the oocyte, and in MIPP, a mouse placental protein.We have determined a 13Å helical 3-dimensional structure of the actin-scruin complex, which is the basic repeat unit of the acrosomal bundle in Limulus sperm and have fitted the F-actin filament of Holmes. Each scruin binds to two adjacent actin molecules along a single F-actin filament helix. In the present investigation, we used our high resolution (7Å) spot scan electron images of ice-embedded acrosomal bundles for further analysis of the actin atomic map and scruin-scruin contacts. Spot-scan images were obtained from the frozen, hydrated acrosomal bundles in a 400 kV electron cryomicroscope.

scholarly journals Actin-binding proteins sensitively mediate F-actin bundle stiffness

2006 ◽  
Vol 5 (9) ◽  
pp. 748-753 ◽  
Author(s):  
Mireille M. A. E. Claessens ◽  
Mark Bathe ◽  
Erwin Frey ◽  
Andreas R. Bausch
Keyword(s):  
Binding Proteins ◽  
Actin Binding ◽  
Actin Binding Proteins ◽  
Actin Bundle

scholarly journals Structural insights into filament recognition by cellular actin markers

2019 ◽  
Author(s):  
Archana Kumari ◽  
Shubham Kesarwani ◽  
Manjunath G Javoor ◽  
Kutti R. Vinothkumar ◽  
Minhajuddin Sirajuddin
Keyword(s):  
High Resolution ◽  
Binding Sites ◽  
Binding Proteins ◽  
Structural Model ◽  
Structural Data ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Electron Cryomicroscopy ◽  
Structural Insights ◽  
Structural Perspective

AbstractCellular studies of filamentous actin (F-actin) processes commonly utilize fluorescent versions of toxins, peptides and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F-actin markers for a particular requirement. Here we describe the electron cryomicroscopy structures of phalloidin, lifeAct and utrophin bound to F-actin, providing the first high-resolution structures and comparison of widely used actin markers and their influence towards F-actin. Our results show that phalloidin binding does not induce conformations and lifeAct specifically recognizes ADP-actin state, which can be used as a sensor for distinguishing different nucleotide states of F-actin. The utrophin structural model aided designing minimal utrophin, which can be utilized as F-actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin binding proteins. Further offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants.

3-D structure of single filaments from the acrosomal bundle of Limulus sperm by 400 kV cryoelectron microscopy: Scruin binds to homologous actin subdomains

1994 ◽  
Vol 52 ◽  
pp. 94-95
Author(s):  
Jacob Agris ◽  
Michael F. Schmid ◽  
Joanita Jakana ◽  
Paul Matsudaira ◽  
Wah Chiu
Keyword(s):  
Actin Binding ◽  
Structural Basis ◽  
Cryoelectron Microscopy ◽  
Ring Canal ◽  
Sequence Comparisons ◽  
3 Dimensional ◽  
Binding Domains ◽  
New Family ◽  
Placental Protein ◽  
Dimensional Crystal

Scruin (102 kDa), an actin binding and bundling protein, is found in a 1:1 complex with actin in the acrosomal process of Limulus sperm. The protein sequence of scruin is unrelated to any known actin binding proteins but reveals a tandem pair of homologous domains (Way and Matsudaira, manuscript submitted). Sequence comparisons reveal homology in each domain with kelch, a gene in Drosophila that is important in nutrient transport into the oocyte and in the maintenance of a cellular structure called the ring canal, and with MIPP, a mouse placental protein of unknown function. Their similarity with scruin suggests a cytoskeletal function and they may form a new family of actin crosslinking proteins. The actin binding domains in this family of proteins have not been identified.To understand the structural basis of actin crosslinking and bundling, we have been studying the acrosomal bundle by cryoelectron microscopy. The actin bundle of the Limulus acrosomal process is structurally unique because it can be considered as a single 3-dimensional crystal in space group PI, with 28 actin subunits and scruin molecules per unit cell.

scholarly journals Structural effects and functional implications of phalloidin and jasplakinolide binding to actin filaments

2019 ◽  
Author(s):  
Sabrina Pospich ◽  
Felipe Merino ◽  
Stefan Raunser
Keyword(s):  
High Resolution ◽  
Actin Filament ◽  
Actin Filaments ◽  
Inorganic Phosphate ◽  
Binding Proteins ◽  
Conformational Space ◽  
Actin Binding ◽  
List Type ◽  
Filamentous Actin ◽  
Actin Binding Proteins

SummaryActin undergoes structural transitions during polymerization, ATP hydrolysis and subsequent release of inorganic phosphate. Several actin binding proteins sense specific states during this transition and can thus target different regions of the actin filament. Here we show in atomic detail that phalloidin, a mushroom toxin that is routinely used to stabilize and label actin filaments, suspends the structural changes in actin, likely influencing its interaction with actin binding proteins. Furthermore, high-resolution cryo-EM structures reveal structural rearrangements in F-actin upon inorganic phosphate release in phalloidin-stabilized filaments. We find that the effect of the sponge toxin jasplakinolide differs from the one of phalloidin, despite their overlapping binding site and similar interactions with the actin filament. Analysis of structural conformations of F-actin suggests that stabilizing agents trap states within the natural conformational space of actin.Abstract FigureHighlightsFive high-resolution cryo-EM structures of stabilized filamentous actinPhalloidin traps different structural states depending on when it is addedThe effect of phalloidin and jasplakinolide on filamentous actin is not identicalBoth toxins likely interfere with the binding of proteins sensing F-actin’s nucleotide state

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