The Spectraplakin Short Stop Is an Actin–Microtubule Cross-Linker That Contributes to Organization of the Microtubule Network

The dynamics of actin and microtubules are coordinated in a variety of cellular and morphogenetic processes; however, little is known about the molecules mediating this cytoskeletal cross-talk. We are studying Short stop (Shot), the sole Drosophila spectraplakin, as a model actin–microtubule cross-linking protein. Spectraplakins are an ancient family of giant cytoskeletal proteins that are essential for a diverse set of cellular functions; yet, we know little about the dynamics of spectraplakins and how they bridge actin filaments and microtubules. In this study we describe the intracellular dynamics of Shot and a structure–function analysis of its role as a cytoskeletal cross-linker. We find that Shot interacts with microtubules using two different mechanisms. In the cell interior, Shot binds growing plus ends through an interaction with EB1. In the cell periphery, Shot associates with the microtubule lattice via its GAS2 domain, and this pool of Shot is actively engaged as a cross-linker via its NH2-terminal actin-binding calponin homology domains. This cross-linking maintains microtubule organization by resisting forces that produce lateral microtubule movements in the cytoplasm. Our results provide the first description of the dynamics of these important proteins and provide key insight about how they function during cytoskeletal cross-talk.

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Migration and bidirectional phototaxis in Dictyostelium discoideum slugs lacking the actin cross-linking 120 kDa gelation factor.

Journal of Experimental Biology ◽

10.1242/jeb.200.24.3213 ◽

1997 ◽

Vol 200 (24) ◽

pp. 3213-3220 ◽

Cited By ~ 1

Author(s):

E Wallraff ◽

H G Wallraff

Keyword(s):

Dictyostelium Discoideum ◽

Incident Light ◽

Actin Binding ◽

Cross Linking ◽

Alternative Possibility ◽

Wild Type ◽

Oblique Angle ◽

Mutant Strains ◽

Cross Linker ◽

Actin Capping

Three mutant strains of Dictyostelium discoideum, lacking different actin-binding proteins, were tested for behavioural deficits in the multicellular pseudoplasmodium (slug) stage. Two strains, defective in the production of either -actinin (an actin cross-linker) or severin (an actin capping and severing protein), did not show changes in slug behaviour. Slugs of the mutant lacking another actin cross-linker, the 120 kDa gelation factor (ABP-120), however, migrated shorter distances in darkness as well as in horizontally directed light. More remarkably, they migrated at an angle of approximately 45 degrees to the left or right of the incident light, whereas wild-type slugs migrated on fairly straight paths towards the light. We discuss the hypothesis that this bidirectional oblique-angle phototaxis is due to changes in the optical properties of the pseudoplasmodia. Normally, in wild-type slugs, a lens effect causes stronger stimulation on the side distal to the incident light. We propose that in the mutant the lens quality is reduced, so that at small angles between the slug axis and the rays of light the proximal side is stimulated more intensely. As a result, the intended symmetrical stimulation is achieved at a certain angle to the left or right of the incident light. We assume that the absence of ABP-120 alters the shape of the lens and/or enhances internal light scattering via degradation of intercellular coherence; however, intracellular attenuation of light remains an additional or alternative possibility.

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The Abl-related gene (Arg) requires its F-actin–microtubule cross-linking activity to regulate lamellipodial dynamics during fibroblast adhesion

The Journal of Cell Biology ◽

10.1083/jcb.200308055 ◽

2004 ◽

Vol 165 (3) ◽

pp. 407-420 ◽

Cited By ~ 76

Author(s):

Ann L. Miller ◽

Yinxiang Wang ◽

Mark S. Mooseker ◽

Anthony J. Koleske

Keyword(s):

Molecular Mechanisms ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Physical Contact ◽

Actin Binding ◽

Cross Linking ◽

Cell Periphery ◽

Related Gene ◽

Filamentous Actin ◽

Protein Fusion

Microtubules (MTs) help establish and maintain cell polarity by promoting actin-dependent membrane protrusion at the leading edge of the cell, but the molecular mechanisms that mediate cross-talk between actin and MTs during this process are unclear. We demonstrate that the Abl-related gene (Arg) nonreceptor tyrosine kinase is required for dynamic lamellipodial protrusions after adhesion to fibronectin. arg−/− fibroblasts exhibit reduced lamellipodial dynamics as compared with wild-type fibroblasts, and this defect can be rescued by reexpression of an Arg-yellow fluorescent protein fusion. We show that Arg can bind MTs with high affinity and cross-link filamentous actin (F-actin) bundles and MTs in vitro. MTs concentrate and insert into Arg-induced F-actin–rich cell protrusions. Arg requires both its F-actin–binding domains and its MT-binding domain to rescue the defects in lamellipodial dynamics of arg−/− fibroblasts. These findings demonstrate that Arg can mediate physical contact between F-actin and MTs at the cell periphery and that this cross-linking activity is required for Arg to regulate lamellipodial dynamics in fibroblasts.

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An Interaction Type of Genetic Screen Reveals a Role of the Rab11 Gene in oskar mRNA Localization in the Developing Drosophila melanogaster Oocyte

Genetics ◽

10.1093/genetics/158.3.1177 ◽

2001 ◽

Vol 158 (3) ◽

pp. 1177-1188

Author(s):

Ferenc Jankovics ◽

Rita Sinka ◽

Miklós Erdélyi

Keyword(s):

Drosophila Melanogaster ◽

Mrna Localization ◽

Posterior Pole ◽

Genetic Screen ◽

Transport Processes ◽

Follicle Cells ◽

Actin Binding ◽

Interaction Type ◽

Microtubule Organization ◽

Microtubule Network

Abstract Abdomen and germ cell development of Drosophila melanogaster embryo requires proper localization of oskar mRNA to the posterior pole of the developing oocyte. oskar mRNA localization depends on complex cell biological events like cell-cell communication, dynamic rearrangement of the microtubule network, and function of the actin cytoskeleton of the oocyte. To investigate the cellular mechanisms involved, we developed a novel interaction type of genetic screen by which we isolated 14 dominant enhancers of a sensitized genetic background composed of mutations in oskar and in TropomyosinII, an actin binding protein. Here we describe the detailed analysis of two allelic modifiers that identify Drosophila Rab11, a gene encoding small monomeric GTPase. We demonstrate that mutation of the Rab11 gene, involved in various vesicle transport processes, results in ectopic localization of oskar mRNA, whereas localization of gurken and bicoid mRNAs and signaling between the oocyte and the somatic follicle cells are unaffected. We show that the ectopic oskar mRNA localization in the Rab11 mutants is a consequence of an abnormally polarized oocyte microtubule cytoskeleton. Our results indicate that the internal membranous structures play an important role in the microtubule organization in the Drosophila oocyte and, thus, in oskar RNA localization.

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The network organization and the phosphorylation of cytokeratins are concomitantly modified by forskolin in the enterocyte-like differentiated Caco-2 cell line

Journal of Cell Science ◽

10.1242/jcs.107.10.2909 ◽

1994 ◽

Vol 107 (10) ◽

pp. 2909-2918 ◽

Cited By ~ 1

Author(s):

L. Baricault ◽

B. de Nechaud ◽

C. Sapin ◽

P. Codogno ◽

P. Denoulet ◽

...

Keyword(s):

Spatial Organization ◽

Phosphorylation Site ◽

Human Colon ◽

Cell System ◽

Cytoskeletal Proteins ◽

Cell Periphery ◽

Type I ◽

Cellular Functions ◽

Morphological Alterations ◽

Continuous Presence

Confluent Caco-2 cells, originating from a human colon carcinoma, display morphological and functional characteristics of differentiated enterocytes such as the presence of a polarized monolayer covered by an apical brush border that express several hydrolases. The adaptation of these cells to grow in the continuous presence of forskolin, a drug known to stimulate adenylyl cyclase permanently, has been previously shown to result in a decreased apical expression of hydrolases and in morphological alterations including the disappearance of intercellular spaces and shortening of microvilli. In the present work we have analyzed the possibility that cytoskeletal proteins may be the target of forskolin in living Caco-2 cells. We show that forskolin initiates dramatic changes in the spatial organization of the cytokeratin network that correlate with an increased phosphorylation of cytokeratin molecules, whereas microtubules, microfilaments and vimentin remain mainly unaffected. Indirect immunofluorescence studies show that the cytokeratin network is redistributed from the cell periphery to the cytoplasm. Biochemical experiments indicate that forskolin doesn't interfere with the cytokeratin profile, since the three cytokeratins normally found in intestine (CK 8, CK 18, CK 19) are similarly expressed in both control and forskolin-Caco-2 cells. Analysis of 32P-labeled cytokeratin extracted from the two cell populations demonstrates that forskolin quantitatively increases the phosphorylation of type I cytokeratin (CK 18 and CK 19), whereas the phosphorylation of type II cytokeratin (CK 8) is altered both quantitatively and qualitatively with the emergence of a new phosphorylation site. These results provide a new cell system in which it is possible to control the subcellular distribution of cytokeratin by changing their phosphorylation status and therefore to study their potential cellular functions.

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Effects of actin filament cross-linking and filament length on actin-myosin interaction.

The Journal of Cell Biology ◽

10.1083/jcb.101.5.1850 ◽

1985 ◽

Vol 101 (5) ◽

pp. 1850-1857 ◽

Cited By ~ 29

Author(s):

T R Coleman ◽

M S Mooseker

Keyword(s):

Atpase Activity ◽

Actin Filament ◽

Actin Filaments ◽

Actin Binding ◽

Molar Ratio ◽

Cross Linking ◽

Filament Length ◽

Maximum Enhancement ◽

Actomyosin Atpase ◽

Cross Linker

We have used two actin-binding proteins of the intestinal brush border, TW 260/240 and villin, to examine the effects of filament cross-linking and filament length on myosin-actin interactions. TW 260/240 is a nonerythroid spectrin that is a potent cross-linker of actin filaments. In the presence of this cross-linker we observed a concentration-dependent enhancement of skeletal muscle actomyosin ATPase activity (150-560% of control; maximum enhancement at a 1:70-80 TW 260/240:actin molar ratio). TW 260/240 did not cause a similar enhancement of either acto-heavy meromyosin (HMM) ATPase or acto-myosin subfragment-one (S1) ATPase. Villin, a Ca2+-dependent filament capping and severing protein of the intestinal microvillus, was used to generate populations of actin filaments of various lengths from less than 20 nm to 2.0 microns; (villin:actin ratios of 1:2 to 1:4,000). The effect of filament length on actomyosin ATPase was biphasic. At villin:actin molar ratios of 1:2-25 actin-activated myosin ATPase activity was inhibited to 20-80% of control values, with maximum inhibition observed at the highest villin:actin ratio. The ATPase activities of acto-HMM and acto-S1 were also inhibited at these short filament lengths. At intermediate filament lengths generated at villin:actin ratios of 1:40-400 (average lengths 0.26-1.1 micron) an enhancement of actomyosin ATPase was observed (130-260% of controls), with a maximum enhancement at average filament lengths of 0.5 micron. The levels of actomyosin ATPase fell off to control values at low concentrations of villin where filament length distributions were almost those of controls. Unlike intact myosin, the actin-activated ATPase of neither HMM nor S1 showed an enhancement at these intermediate actin filament lengths.

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Super-Resolution Microscopy Reveals That Stromal Interaction Molecule 1 Trafficking Depends on Microtubule Dynamics

Frontiers in Physiology ◽

10.3389/fphys.2021.762387 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yi-Ting Huang ◽

Ya-Ting Hsu ◽

Yih-Fung Chen ◽

Meng-Ru Shen

Keyword(s):

Cell Migration ◽

Cancer Cells ◽

Real World ◽

Cancer Metastasis ◽

Super Resolution ◽

Cell Periphery ◽

Cellular Functions ◽

Microtubule Network ◽

Stromal Interaction Molecule 1 ◽

The Impact

Store-operated Ca2+ entry (SOCE) is an essential pathway for Ca2+ signaling, and regulates various vital cellular functions. It is triggered by the endoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1). Illustration of STIM1 spatiotemporal structure at the nanometer scale during SOCE activation provides structural and functional insights into the fundamental Ca2+ homeostasis. In this study, we used direct stochastic optical reconstruction microscopy (dSTORM) to revisit the dynamic process of the interaction between STIM1, end-binding protein (EB), and microtubules to the ER-plasma membrane. Using dSTORM, we found that“powder-like”STIM1 aggregates into “trabecular-like” architectures toward the cell periphery during SOCE, and that an intact microtubule network and EB1 are essential for STIM1 trafficking. After thapsigargin treatment, STIM1 can interact with EB1 regardless of undergoing aggregation. We generated STIM1 variants adapted from a real-world database and introduced them into SiHa cells to clarify the impact of STIM1 mutations on cancer cell behavior. The p.D76G and p.D84Y variants locating on the Ca2+ binding domain of STIM1 result in inhibition of focal adhesion turnover, Ca2+ influx during SOCE and subsequent cell migration. Inversely, the p.R643C variant on the microtubule interacting domain of STIM1 leads to dissimilar consequence and aggravates cell migration. These findings imply that STIM1 mutational patterns have an impact on cancer metastasis, and therefore could be either a prognostic marker or a novel therapeutic target to inhibit the malignant behavior of STIM1-mediated cancer cells. Altogether, we generated novel insight into the role of STIM1 during SOCE activation, and uncovered the impact of real-world STIM1 variants on cancer cells.

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The actin regulator profilin 1 is functionally associated with the mammalian centrosome

Life Science Alliance ◽

10.26508/lsa.202000655 ◽

2020 ◽

Vol 4 (1) ◽

pp. e202000655

Author(s):

Michaela Nejedlá ◽

Anastasiya Klebanovych ◽

Vadym Sulimenko ◽

Tetyana Sulimenko ◽

Eduarda Dráberová ◽

...

Keyword(s):

Actin Polymerization ◽

Binding Proteins ◽

Actin Binding ◽

Cell Periphery ◽

Microtubule Nucleation ◽

Microtubule Organization ◽

Actin Binding Proteins ◽

Cell Edge ◽

It Impacts ◽

Microtubule Growth

Profilin 1 is a crucial actin regulator, interacting with monomeric actin and several actin-binding proteins controlling actin polymerization. Recently, it has become evident that this profilin isoform associates with microtubules via formins and interferes with microtubule elongation at the cell periphery. Recruitment of microtubule-associated profilin upon extensive actin polymerizations, for example, at the cell edge, enhances microtubule growth, indicating that profilin contributes to the coordination of actin and microtubule organization. Here, we provide further evidence for the profilin-microtubule connection by demonstrating that it also functions in centrosomes where it impacts on microtubule nucleation.

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The stable actin core of mechanosensory stereocilia features continuous turnover of actin cross-linkers

Molecular Biology of the Cell ◽

10.1091/mbc.e18-03-0196 ◽

2018 ◽

Vol 29 (15) ◽

pp. 1856-1865 ◽

Cited By ~ 4

Author(s):

Pallabi Roy ◽

Benjamin J. Perrin

Keyword(s):

Hair Cell ◽

Hair Cells ◽

Head Movement ◽

Actin Filaments ◽

Actin Dynamics ◽

Actin Binding ◽

Cross Linking ◽

Sensory Hair Cells ◽

Actin Turnover ◽

Cross Linker

Stereocilia are mechanosensitive protrusions on the surfaces of sensory hair cells in the inner ear that detect sound, gravity, and head movement. Their cores are composed of parallel actin filaments that are cross-linked and stabilized by several actin-binding proteins, including fascin-2, plastin-1, espin, and XIRP2. The actin filaments are the most stable known, with actin turnover primarily occurring at the stereocilia tips. While stereocilia actin dynamics has been well studied, little is known about the behavior of the actin cross-linking proteins, which are the most abundant type of protein in stereocilia after actin and are critical for stereocilia morphogenesis and maintenance. Here, we developed a novel transgenic mouse to monitor EGFP-fascin-2 incorporation . In contrast to actin, EGFP-fascin-2 readily enters the stereocilia core. We also compared the effect of EGFP-fascin-2 expression on developing and mature stereocilia. When it was induced during hair cell development, we observed increases in both stereocilia length and width. Interestingly, stereocilia size was not affected when EGFP-fascin-2 was induced in adult stereocilia. Regardless of the time of induction, EGFP-fascin-2 displaced both espin and plastin-1 from stereocilia. Altering the actin cross-linker composition, even as the actin filaments exhibit little to no turnover, provides a mechanism for ongoing remodeling and repair important for stereocilia homeostasis.

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Effect of the Cross-Linking Density on the Swelling and Rheological Behavior of Ester-Bridged β-Cyclodextrin Nanosponges

Materials ◽

10.3390/ma14030478 ◽

2021 ◽

Vol 14 (3) ◽

pp. 478

Author(s):

Gjylije Hoti ◽

Fabrizio Caldera ◽

Claudio Cecone ◽

Alberto Rubin Pedrazzo ◽

Anastasia Anceschi ◽

...

Keyword(s):

Scale Up ◽

Future Application ◽

Cross Linking ◽

Synthesis Reaction ◽

Dependent Behavior ◽

Cyclodextrin Nanosponges ◽

Potential Applications ◽

Cross Linker ◽

The Cross ◽

Shed Light

The cross-linking density influences the physicochemical properties of cyclodextrin-based nanosponges (CD-NSs). Although the effect of the cross-linker type and content on the NSs performance has been investigated, a detailed study of the cross-linking density has never been performed. In this contribution, nine ester-bridged NSs based on β-cyclodextrin (β-CD) and different quantities of pyromellitic dianhydride (PMDA), used as a cross-linking agent in stoichiometric proportions of 2, 3, 4, 5, 6, 7, 8, 9, and 10 moles of PMDA for each mole of CD, were synthesized and characterized in terms of swelling and rheological properties. The results, from the swelling experiments, exploiting Flory–Rehner theory, and rheology, strongly showed a cross-linker content-dependent behavior. The study of cross-linking density allowed to shed light on the efficiency of the synthesis reaction methods. Overall, our study demonstrates that by varying the amount of cross-linking agent, the cross-linked structure of the NSs matrix can be controlled effectively. As PMDA βCD-NSs have emerged over the years as a highly versatile class of materials with potential applications in various fields, this study represents the first step towards a full understanding of the correlation between their structure and properties, which is a key requirement to effectively tune their synthesis reaction in view of any specific future application or industrial scale-up.

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FLIm and Raman Spectroscopy for Investigating Biochemical Changes of Bovine Pericardium upon Genipin Cross-Linking

Molecules ◽

10.3390/molecules25173857 ◽

2020 ◽

Vol 25 (17) ◽

pp. 3857

Author(s):

Tanveer Ahmed Shaik ◽

Alba Alfonso-Garcia ◽

Martin Richter ◽

Florian Korinth ◽

Christoph Krafft ◽

...

Keyword(s):

Raman Spectroscopy ◽

Fluorescence Lifetime ◽

Density Functional ◽

Bovine Pericardium ◽

Biochemical Changes ◽

Blue Pigment ◽

Cross Linking ◽

Fluorescence Lifetime Imaging ◽

Fluorescence Intensity Ratio ◽

Cross Linker

Biomaterials used in tissue engineering and regenerative medicine applications benefit from longitudinal monitoring in a non-destructive manner. Label-free imaging based on fluorescence lifetime imaging (FLIm) and Raman spectroscopy were used to monitor the degree of genipin (GE) cross-linking of antigen-removed bovine pericardium (ARBP) at three incubation time points (0.5, 1.0, and 2.5 h). Fluorescence lifetime decreased and the emission spectrum redshifted compared to that of uncross-linked ARBP. The Raman signature of GE-ARBP was resonance-enhanced due to the GE cross-linker that generated new Raman bands at 1165, 1326, 1350, 1380, 1402, 1470, 1506, 1535, 1574, 1630, 1728, and 1741 cm−1. These were validated through density functional theory calculations as cross-linker-specific bands. A multivariate multiple regression model was developed to enhance the biochemical specificity of FLIm parameters fluorescence intensity ratio (R2 = 0.92) and lifetime (R2 = 0.94)) with Raman spectral results. FLIm and Raman spectroscopy detected biochemical changes occurring in the collagenous tissue during the cross-linking process that were characterized by the formation of a blue pigment which affected the tissue fluorescence and scattering properties. In conclusion, FLIm parameters and Raman spectroscopy were used to monitor the degree of cross-linking non-destructively.

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