scholarly journals Genetic deletion of ABP-120 alters the three-dimensional organization of actin filaments in Dictyostelium pseudopods.

1995 ◽  
Vol 128 (5) ◽  
pp. 819-835 ◽  
Author(s):  
D Cox ◽  
J A Ridsdale ◽  
J Condeelis ◽  
J Hartwig
Keyword(s):  
Actin Filaments ◽  
Biochemical Analysis ◽  
Three Dimensional ◽  
Cross Linking ◽  
Genetic Deletion ◽  
Triton X ◽  
Filament Network ◽  
Camp Stimulation ◽  
Confocal And Electron Microscopy

This study extends the observations on the defects in pseudopod formation of ABP-120+ and ABP-120- cells by a detailed morphological and biochemical analysis of the actin based cytoskeleton. Both ABP-120+ and ABP-120- cells polymerize the same amount of F-actin in response to stimulation with cAMP. However, unlike ABP-120+ cells, ABP-120- cells do not incorporate actin into the Triton X-100-insoluble cytoskeleton at 30-50 s, the time when ABP-120 is incorporated into the cytoskeleton and when pseudopods are extended after cAMP stimulation in wild-type cells. By confocal and electron microscopy, pseudopods extended by ABP-120- cells are not as large or thick as those produced by ABP-120+ cells and in the electron microscope, an altered filament network is found in pseudopods of ABP-120- cells when compared to pseudopods of ABP-120+ cells. The actin filaments found in areas of pseudopods in ABP-120+ cells either before or after stimulation were long, straight, and arranged into space filling orthogonal networks. Protrusions of ABP-120- cells are less three-dimensional, denser, and filled with multiple foci of aggregated filaments consistent with collapse of the filament network due to the absence of ABP-120-mediated cross-linking activity. The different organization of actin filaments may account for the diminished size of protrusions observed in living and fixed ABP-120- cells compared to ABP-120+ cells and is consistent with the role of ABP-120 in regulating pseudopod extension through its cross-linking of actin filaments.

Televisualization: An Aid To Collaborative Research In Molecular Structure Biology

1998 ◽  
Vol 4 (S2) ◽  
pp. 32-33
Author(s):  
M. F. Schmid ◽  
P. Matsudaira ◽  
M. T. Dougherty ◽  
M. B. Sherman ◽  
C. Henn ◽  
...  
Keyword(s):  
Image Processing ◽  
Actin Filaments ◽  
Collaborative Research ◽  
Horseshoe Crab ◽  
Hexagonal Lattice ◽  
Three Dimensional ◽  
Limulus Polyphemus ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Tilt Series

Collaboration between local microscopists and image processing specialists, and their remote biological colleagues, has been hampered by the difficulty of i) transferring the three-dimensional reconstructions of macromolecules resulting from the cryomicroscopy and image processing, ii) viewing the results in a meaningful way, and iii) communicating the results and the interpretations derived therefrom to each other.The acrosomal process is an intracellular quasi-crystalline organelle in the head of the sperm of the horseshoe crab Limulus polyphemus. It consists of 100 - 130 actin-scruin filaments packed together in a pseudo-hexagonal lattice and is up to 60 μm long with a diameter of 0.1 μm. Scruin-scruin interactions are responsible for cross-linking the actin filaments together in the bundle. Our goal was to reveal interfilament interactions in the bundle. We have taken tilt series images in the electron microscope to reconstruct its three-dimensional structure at 45 Å resolution.

scholarly journals Targeted disruption of the ABP-120 gene leads to cells with altered motility.

1992 ◽  
Vol 116 (4) ◽  
pp. 943-955 ◽  
Author(s):  
D Cox ◽  
J Condeelis ◽  
D Wessels ◽  
D Soll ◽  
H Kern ◽  
...  
Keyword(s):  
Cell Lines ◽  
Actin Filaments ◽  
Mutant Cell ◽  
Actin Binding ◽  
Cross Linking ◽  
Targeted Disruption ◽  
Before And After ◽  
Expansion Model ◽  
Transformation Vectors ◽  
Camp Stimulation

The actin-binding protein ABP-120 has been proposed to play a role in cross-linking F-actin filaments during pseudopod formation in motile Dictyostelium amebas. We have tested this hypothesis by analyzing the phenotype of mutant cell lines which do not produce ABP-120. Two different transformation vectors capable of targeted disruption of the ABP-120 gene locus have been constructed using a portion of an ABP-120 cDNA clone. Three independent cell lines with different disruption events have been obtained after transformation of amebas with these vectors. The disruption of the ABP-120 gene by vector sequences results in either the production of a small amount of truncated ABP-120 or no detectable protein at all. The phenotypes of two different clones lacking ABP-120, generated in strains AX3 and AX4, have been characterized and show identical results. ABP-120- cells tend to remain rounder before and after cAMP stimulation, and do not reextend pseudopods normally after rapid addition of cAMP. In addition, ABP-120- cells translocating in buffer exhibit defects in both the rate and extent of pseudopod formation. The amount of F-actin cross-linked into the cytoskeleton after cAMP stimulation of ABP-120- cells is reduced at times when ABP-120 has been shown to be incorporated into the cytoskeleton, and this correlates temporally with the absence of reextension of pseudopods after cAMP stimulation. The instantaneous velocity is significantly reduced both before and after cAMP stimulation in the ABP-120- cells, and the cells show decreased chemotactic efficiency compared to ABP-120+ controls. This phenotype is consistent with a role for ABP-120 in pseudopod extension by cross-linking actin filaments as proposed by the "cortical expansion model" (Condeelis, J., A. Bresnick, M. Demma, C. Dharmawardhane, R. Eddy, A. L. Hall, R. Sauterer, and V. Warren. 1990. Dev. Genet. 11:333-340).

Nesprin-3: a versatile connector between the nucleus and the cytoskeleton

2011 ◽  
Vol 39 (6) ◽  
pp. 1719-1724 ◽  
Author(s):  
Mirjam Ketema ◽  
Arnoud Sonnenberg
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Actin Filaments ◽  
Structural Integrity ◽  
Nuclear Lamina ◽  
Cross Linking ◽  
Indirect Interaction ◽  
Direct Link ◽  
Nuclear Interior

The cytoskeleton is connected to the nuclear interior by LINC (linker of nucleoskeleton and cytoskeleton) complexes located in the nuclear envelope. These complexes consist of SUN proteins and nesprins present in the inner and outer nuclear membrane respectively. Whereas SUN proteins can bind the nuclear lamina, members of the nesprin protein family connect the nucleus to different components of the cytoskeleton. Nesprin-1 and -2 can establish a direct link with actin filaments, whereas nesprin-4 associates indirectly with microtubules through its interaction with kinesin-1. Nesprin-3 is the only family member known that can link the nuclear envelope to intermediate filaments. This indirect interaction is mediated by the binding of nesprin-3 to the cytoskeletal linker protein plectin. Furthermore, nesprin-3 can connect the nucleus to microtubules by its interactions with BPAG1 (bullous pemphigoid antigen 1) and MACF (microtubule–actin cross-linking factor). In contrast with the active roles that nesprin-1, -2 and -4 have in actin- and microtubule-dependent nuclear positioning, the role of nesprin-3 is likely to be more passive. We suggest that it helps to stabilize the anchorage of the nucleus within the cytoplasm and maintain the structural integrity and shape of the nucleus.

scholarly journals Interfaces with Structure Dynamics of the Workhorses from Cells Revealed through Cross-Linking Mass Spectrometry (CLMS)

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 382 ◽  
Author(s):  
Umesh Kalathiya ◽  
Monikaben Padariya ◽  
Jakub Faktor ◽  
Etienne Coyaud ◽  
Javier A. Alfaro ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Structural Proteomics ◽  
Cross Linking ◽  
Diverse Range ◽  
Interaction Sites ◽  
Structural Systems Biology ◽  
Rna Complexes

The fundamentals of how protein–protein/RNA/DNA interactions influence the structures and functions of the workhorses from the cells have been well documented in the 20th century. A diverse set of methods exist to determine such interactions between different components, particularly, the mass spectrometry (MS) methods, with its advanced instrumentation, has become a significant approach to analyze a diverse range of biomolecules, as well as bring insights to their biomolecular processes. This review highlights the principal role of chemistry in MS-based structural proteomics approaches, with a particular focus on the chemical cross-linking of protein–protein/DNA/RNA complexes. In addition, we discuss different methods to prepare the cross-linked samples for MS analysis and tools to identify cross-linked peptides. Cross-linking mass spectrometry (CLMS) holds promise to identify interaction sites in larger and more complex biological systems. The typical CLMS workflow allows for the measurement of the proximity in three-dimensional space of amino acids, identifying proteins in direct contact with DNA or RNA, and it provides information on the folds of proteins as well as their topology in the complexes. Principal CLMS applications, its notable successes, as well as common pipelines that bridge proteomics, molecular biology, structural systems biology, and interactomics are outlined.

Crystallographic Reconstruction of the Acrosomal Process from Limulus Polyphemus Sperm

1998 ◽  
Vol 4 (S2) ◽  
pp. 460-461
Author(s):  
M. B. Sherman ◽  
J. Jakana ◽  
S. Sun ◽  
P. Matsudaira ◽  
W. Chiu ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Horseshoe Crab ◽  
Carbon Film ◽  
Three Dimensional ◽  
Ccd Camera ◽  
Limulus Polyphemus ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Diffraction Mode ◽  
Tilt Series

The acrosomal process is an intracellular quasi-crystalline organelle in the head of the sperm of the horseshoe crab Limulus polyphemus. It consists of 100 - 130 actin-scruin filaments packed together in a pseudo-hexagonal lattice2 and is up to 60 (im long with a diameter of 0.1 μm. Scruin-scruin interactions are responsible for cross-linking the actin filaments together in the bundle. The goal of the current study is to reveal interfilament interactions in the bundle. We have taken tilt series images in the electron microscope3 to reconstruct its three-dimensional structure without assuming helical symmetry.The acrosomal process was purified as described.34 Bundles were embedded in vitreous ice over holes on a holey carbon film on copper grids. The specimen was kept at -167°C in a JEOL 4000EX electron microscope operating at 400 kV. Straight 6-10 (im long bundles were found using a TV-rate CCD camera in defocused diffraction mode.

scholarly journals Unravelling the Encapsulation of DNA and Other Biomolecules in HAp Microcalcifications of Human Breast Cancer Tissues by Raman Imaging

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2658
Author(s):  
Monica Marro ◽  
Anna Maria Rodríguez-Rivero ◽  
Cuauhtémoc Araujo-Andrade ◽  
Maria Teresa Fernández-Figueras ◽  
Laia Pérez-Roca ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Mammography Screening ◽  
Human Breast Cancer ◽  
Biochemical Analysis ◽  
Three Dimensional ◽  
Breast Cancer Tissue ◽  
Cancer Tissue ◽  
Human Breast ◽  
Cancer Tissues

Microcalcifications are detected through mammography screening and, depending on their morphology and distribution (BI-RADS classification), they can be considered one of the first indicators of suspicious cancer lesions. However, the formation of hydroxyapatite (HAp) calcifications and their relationship with malignancy remains unknown. In this work, we report the most detailed three-dimensional biochemical analysis of breast cancer microcalcifications to date, combining 3D Raman spectroscopy imaging and advanced multivariate analysis in order to investigate in depth the molecular composition of HAp calcifications found in 26 breast cancer tissue biopsies. We demonstrate that DNA has been naturally adsorbed and encapsulated inside HAp microcalcifications. Furthermore, we also show the encapsulation of other relevant biomolecules in HAp calcifications, such as lipids, proteins, cytochrome C and polysaccharides. The demonstration of natural DNA biomineralization, particularly in the tumor microenvironment, represents an unprecedented advance in the field, as it can pave the way to understanding the role of HAp in malignant tissues.

scholarly journals Actin mutations that show suppression with fimbrin mutations identify a likely fimbrin-binding site on actin.

1994 ◽  
Vol 126 (2) ◽  
pp. 413-422 ◽  
Author(s):  
J E Honts ◽  
T S Sandrock ◽  
S M Brower ◽  
J L O'Dell ◽  
A E Adams
Keyword(s):  
Crystal Structure ◽  
Sequence Analysis ◽  
Binding Site ◽  
Actin Filaments ◽  
Biochemical Analysis ◽  
Actin Binding ◽  
Cross Linking ◽  
Temperature Sensitive ◽  
Small Domain

Actin interacts with a large number of different proteins that modulate its assembly and mediate its functions. One such protein is the yeast actin-binding protein Sac6p, which is homologous to vertebrate fimbrin (Adams, A. E. M., D. Botstein, and D. G. Drubin. 1991. Nature (Lond.). 354:404-408.). Sac6p was originally identified both genetically (Adams, A. E. M., and D. Botstein. 1989. Genetics. 121:675-683.) by dominant, reciprocal suppression of a temperature-sensitive yeast actin mutation (act1-1), as well as biochemically (Drubin, D. G., K. G. Miller, and D. Botstein. 1988. J. Cell Biol. 107: 2551-2561.). To identify the region on actin that interacts with Sac6p, we have analyzed eight different act1 mutations that show suppression with sac6 mutant alleles, and have asked whether (a) these mutations occur in a small defined region on the crystal structure of actin; and (b) the mutant actins are defective in their interaction with Sac6p in vitro. Sequence analysis indicates that all of these mutations change residues that cluster in the small domain of the actin crystal structure, suggesting that this region is an important part of the Sac6p-binding domain. Biochemical analysis reveals defects in the ability of several of the mutant actins to bind Sac6p, and a reduction in Sac6p-induced cross-linking of mutant actin filaments. Together, these observations identify a likely site of interaction of fimbrin on actin.

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

2019 ◽  
Vol 116 (17) ◽  
pp. 8326-8335 ◽  
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.

scholarly journals Role of fascin in filopodial protrusion

2006 ◽  
Vol 174 (6) ◽  
pp. 863-875 ◽  
Author(s):  
Danijela Vignjevic ◽  
Shin-ichiro Kojima ◽  
Yvonne Aratyn ◽  
Oana Danciu ◽  
Tatyana Svitkina ◽  
...  
Keyword(s):  
Rna Interference ◽  
Dynamic Behavior ◽  
Actin Filaments ◽  
Cellular Localization ◽  
Fluorescence Recovery After Photobleaching ◽  
Cross Linking ◽  
Wild Type ◽  
Actin Organization ◽  
Cross Linker

In this study, the mechanisms of actin-bundling in filopodia were examined. Analysis of cellular localization of known actin cross-linking proteins in mouse melanoma B16F1 cells revealed that fascin was specifically localized along the entire length of all filopodia, whereas other actin cross-linkers were not. RNA interference of fascin reduced the number of filopodia, and remaining filopodia had abnormal morphology with wavy and loosely bundled actin organization. Dephosphorylation of serine 39 likely determined cellular filopodia frequency. The constitutively active fascin mutant S39A increased the number and length of filopodia, whereas the inactive fascin mutant S39E reduced filopodia frequency. Fluorescence recovery after photobleaching of GFP-tagged wild-type and S39A fascin showed that dephosphorylated fascin underwent rapid cycles of association to and dissociation from actin filaments in filopodia, with t1/2 < 10 s. We propose that fascin is a key specific actin cross-linker, providing stiffness for filopodial bundles, and that its dynamic behavior allows for efficient coordination between elongation and bundling of filopodial actin filaments.

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

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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