Cortical instability drives periodic supracellular actin pattern formation in epithelial tubes

An essential question of morphogenesis is how patterns arise without preexisting positional information, as inspired by Turing. In the past few years, cytoskeletal flows in the cell cortex have been identified as a key mechanism of molecular patterning at the subcellular level. Theoretical and in vitro studies have suggested that biological polymers such as actomyosin gels have the property to self-organize, but the applicability of this concept in an in vivo setting remains unclear. Here, we report that the regular spacing pattern of supracellular actin rings in the Drosophila tracheal tubule is governed by a self-organizing principle. We propose a simple biophysical model where pattern formation arises from the interplay of myosin contractility and actin turnover. We validate the hypotheses of the model using photobleaching experiments and report that the formation of actin rings is contractility dependent. Moreover, genetic and pharmacological perturbations of the physical properties of the actomyosin gel modify the spacing of the pattern, as the model predicted. In addition, our model posited a role of cortical friction in stabilizing the spacing pattern of actin rings. Consistently, genetic depletion of apical extracellular matrix caused strikingly dynamic movements of actin rings, mirroring our model prediction of a transition from steady to chaotic actin patterns at low cortical friction. Our results therefore demonstrate quantitatively that a hydrodynamical instability of the actin cortex can trigger regular pattern formation and drive morphogenesis in an in vivo setting.

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Vimentin provides the mechanical resilience required for amoeboid migration and protection of the nucleus

10.1101/720946 ◽

2019 ◽

Cited By ~ 4

Author(s):

Luiza Da Cunha Stankevicins ◽

Marta Urbanska ◽

Daniel AD. Flormann ◽

Emmanuel Terriac ◽

Zahra Mostajeran ◽

...

Keyword(s):

Dna Damage ◽

Dendritic Cells ◽

Cell Migration ◽

Molecular Mechanisms ◽

Cell Cortex ◽

Dna Double Strand Breaks ◽

Nuclear Positioning ◽

Mechanical Stiffness

AbstractDendritic cells use amoeboid migration through constricted passages to reach the lymph nodes, and this homing function is crucial for immune responses. Amoeboid migration requires mechanical resilience, however, the underlying molecular mechanisms for this type of migration remain unknown. Because vimentin intermediate filaments (IFs) and microfilaments regulate adhesion-dependent migration in a bidirectional manner, we analyzed if they exert a similar control on amoeboid migration. Vimentin was required for cellular resilience, via a joint interaction between vimentin IFs and F-actin. Reduced actin mobility in the cell cortex of vimentin-reduced cells indicated that vimentin promotes Factin subunit exchange and dynamics. These mechano-dynamic alterations in vimentin-deficient dendritic cells impaired amoeboid migration in confined environments in vitro and blocked lymph node homing in mouse experiments in vivo. Correct nuclear positioning is important in confined amoeboid migration both to minimize resistance and to avoid DNA damage. Vimentin-deficiency also led to DNA double strand breaks in the compressed dendritic cells, pointing to a role of vimentin in nuclear positioning. Together, these observations show that vimentin IF-microfilament interactions provide both the specific mechano-dynamics required for dendritic cell migration and the protection the genome needs in compressed spaces.Summary statementVimentin — in joint action with actin — mediates the mechanical stiffness of cells required for amoeboid cell migration through confined spaces and protects the nucleus from DNA damage.

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Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex

eLife ◽

10.7554/elife.10785 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 44

Author(s):

Andrei Vovk ◽

Chad Gu ◽

Michael G Opferman ◽

Larisa E Kapinos ◽

Roderick YH Lim ◽

...

Keyword(s):

Spatial Organization ◽

Intrinsically Disordered Proteins ◽

Nucleocytoplasmic Transport ◽

Transport Proteins ◽

Disordered Proteins ◽

Biophysical Model ◽

Nuclear Pore ◽

Intrinsically Disordered

Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function.

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Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements

Development ◽

10.1242/dev.125.22.4349 ◽

1998 ◽

Vol 125 (22) ◽

pp. 4349-4358 ◽

Cited By ~ 4

Author(s):

J. Charite ◽

W. de Graaff ◽

D. Consten ◽

M.J. Reijnen ◽

J. Korving ◽

...

Keyword(s):

Binding Sites ◽

Hox Genes ◽

Regulatory Element ◽

Ectopic Expression ◽

Positional Information ◽

Regulatory Elements ◽

Gene Products ◽

Anteroposterior Patterning

Studies of pattern formation in the vertebrate central nervous system indicate that anteroposterior positional information is generated in the embryo by signalling gradients of an as yet unknown nature. We searched for transcription factors that transduce this information to the Hox genes. Based on the assumption that the activity levels of such factors might vary with position along the anteroposterior axis, we devised an in vivo assay to detect responsiveness of cis-acting sequences to such differentially active factors. We used this assay to analyze a Hoxb8 regulatory element, and detected the most pronounced response in a short stretch of DNA containing a cluster of potential CDX binding sites. We show that differentially expressed DNA binding proteins are present in gastrulating embryos that bind to these sites in vitro, that cdx gene products are among these, and that binding site mutations that abolish binding of these proteins completely destroy the ability of the regulatory element to drive regionally restricted expression in the embryo. Finally, we show that ectopic expression of cdx gene products anteriorizes expression of reporter transgenes driven by this regulatory element, as well as that of the endogenous Hoxb8 gene, in a manner that is consistent with them being essential transducers of positional information. These data suggest that, in contrast to Drosophila Caudal, vertebrate cdx gene products transduce positional information directly to the Hox genes, acting through CDX binding sites in their enhancers. This may represent the ancestral mode of action of caudal homologues, which are involved in anteroposterior patterning in organisms with widely divergent body plans and modes of development.

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SM22α inhibits lamellipodium formation and migration via Ras-Arp2/3 signaling in synthetic VSMCs

AJP Cell Physiology ◽

10.1152/ajpcell.00033.2016 ◽

2016 ◽

Vol 311 (5) ◽

pp. C758-C767 ◽

Cited By ~ 10

Author(s):

Pin Lv ◽

Fan Zhang ◽

Ya-Juan Yin ◽

Yu-Can Wang ◽

Min Gao ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Lesion ◽

Flavonoid Compound ◽

Cell Cortex ◽

Dependent Manner ◽

Nucleotide Exchange ◽

Migration Activity ◽

And Migration

We previously demonstrated that smooth muscle (SM) 22α promotes the migration activity in contractile vascular smooth muscle cells (VSMCs). Based on the varied functions exhibited by SM22α in different VSMC phenotypes, we investigated the effect of SM22α on VSMC migration under pathological conditions. The results demonstrated that SM22α overexpression in synthetic VSMCs inhibited platelet-derived growth factor (PDGF)-BB-induced cell lamellipodium formation and migration, which was different from its action in contractile cells. The results indicated two distinct mechanisms underlying inhibition of lamellipodium formation by SM22α, increased actin dynamic stability and decreased Ras activity via interference with interactions between Ras and guanine nucleotide exchange factor. The former inhibited actin cytoskeleton rearrangement in the cell cortex, while the latter significantly disrupted actin nucleation activation of the Arp2/3 complex. Baicalin, a herb-derived flavonoid compound, inhibited VSMC migration via upregulation of SM22α expression in vitro and in vivo. These data suggest that SM22α regulates lamellipodium formation and cell migration in a phenotype-dependent manner in VSMCs, which may be a new therapeutic target for vascular lesion formation.

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Functional insight into the role of Orc6 in septin complex filament formation in Drosophila

Molecular Biology of the Cell ◽

10.1091/mbc.e14-02-0734 ◽

2015 ◽

Vol 26 (1) ◽

pp. 15-28 ◽

Cited By ~ 19

Author(s):

Katarina Akhmetova ◽

Maxim Balasov ◽

Richard P. H. Huijbregts ◽

Igor Chesnokov

Keyword(s):

Origin Recognition Complex ◽

Spatial Organization ◽

Cell Cortex ◽

Filament Formation ◽

Gtp Binding ◽

Complex Functions ◽

Septin Filament

Septins belong to a family of polymerizing GTP-binding proteins that are important for cytokinesis and other processes that involve spatial organization of the cell cortex. We reconstituted a recombinant Drosophila septin complex and compared activities of the wild-type and several mutant septin complex variants both in vitro and in vivo. We show that Drosophila septin complex functions depend on the intact GTP-binding and/or hydrolysis domains of Pnut, Sep1, and Sep2. The presence of the functional C-terminal domain of septins is required for the integrity of the complex. Drosophila Orc6 protein, the smallest subunit of the origin recognition complex (ORC), directly binds to septin complex and facilitates septin filament formation. Orc6 forms dimers through the interactions of its N-terminal, TFIIB-like domains. This ability of the protein suggests a direct bridging role for Orc6 in stimulating septin polymerization in Drosophila. Studies reported here provide a functional dissection of a Drosophila septin complex and highlight the basic conserved and divergent features among metazoan septin complexes.

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Accurate and sensitive quantification of protein-DNA binding affinity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1714376115 ◽

2018 ◽

Vol 115 (16) ◽

pp. E3692-E3701 ◽

Cited By ~ 32

Author(s):

Chaitanya Rastogi ◽

H. Tomas Rube ◽

Judith F. Kribelbauer ◽

Justin Crocker ◽

Ryan E. Loker ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Binding Sites ◽

Biophysical Model ◽

Regulatory Sequences ◽

Binding Modes ◽

Perfect Agreement ◽

Dna Binding Sites

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes.

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Active ribosome profiling with RiboLace

10.1101/179671 ◽

2017 ◽

Author(s):

Massimiliano Clamer ◽

Toma Tebaldi ◽

Fabio Lauria ◽

Paola Bernabò ◽

Rodolfo F. Gómez-Biagi ◽

...

Keyword(s):

Large Scale ◽

Positional Information ◽

Ribosome Profiling ◽

Input Material ◽

Nuclease Digestion ◽

Rna Fragments ◽

Nucleotide Resolution ◽

Active Ribosome

Ribosome profiling, or Ribo-Seq, is based around large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information concerning ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-Seq approaches lack the ability to distinguish between fragments protected by ribosomes in active translation or by inactive ribosomes. To overcome these significant limitation, we developed RiboLace: a novel method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied bothin vitroandin vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.

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5-hydroxytryptamine-stimulated mitochondrial movement and microvillar growth in the lower malpighian tubule of the insect, Rhodnius prolixus

Journal of Cell Science ◽

10.1242/jcs.49.1.139 ◽

1981 ◽

Vol 49 (1) ◽

pp. 139-161 ◽

Cited By ~ 1

Author(s):

T.J. Bradley ◽

P. Satir

Keyword(s):

Ion Transport ◽

Malpighian Tubule ◽

Fold Increase ◽

Rhodnius Prolixus ◽

Ultrastructural Changes ◽

Cell Cortex ◽

Mitochondrial Movement ◽

The Core

Rapid initiation of ion transport occurs in the lower Malpighian tubule of the insect Rhodnius prolixus following feeding in vivo or stimulation with 5-hydroxytryptamine (5-HT) in vitro. Using the electron microscope, we have conducted a morphometric analysis of cells in the lowest one-third of the lower tubule, demonstrating that 5-HT also induces mitochondrial movement and microvillar growth simultaneously with, but independent of, the onset of ion transport. Mitochondria move from a position below the cell cortex to one inside the microvilli within 10 min of stimulation with 5-HT, resulting in an 8- to 10-fold increase in the volume of mitochondria within the microvilli. Previous findings indicated that mitochondrial movement is dependent on actin-containing microfilaments, but not microtubules. As the mitochondria enter the microvillus, the core microfilaments are reorganized into a sheath of microfilaments, which extends closely parallel to the outer mitochondrial membrane down into the cell interior. This sheath of microfilaments is also observed around mitochondria in the axopods. We suggest that the core microfilaments are responsible for mitochondrial movement into the microvilli and axopods. Stimulation with 5-HT induces a shift in mitochondrial configuration from orthodox to condensed, indicating a possible increase in oxidative phosphorylation. Following stimulation, the microvilli grow about 3 X in volume and 2.5 X in surface area. These increases are more than can be accounted for by mitochondrial invasion and must involve the addition of new membrane and microfilament polymerization. The observed changes - microvillar growth, insertion of additional membrane, activation and movement of mitochondria adjacent to the ion transport membrane - are described in the light of their significance in ion transport. A simple model is proposed which unifies the observed ultrastructural changes and known ion movements in the lower tubule.

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Phosphorylation-site mutagenesis of the growth-associated protein GAP-43 modulates its effects on cell spreading and morphology.

The Journal of Cell Biology ◽

10.1083/jcb.120.2.503 ◽

1993 ◽

Vol 120 (2) ◽

pp. 503-512 ◽

Cited By ~ 71

Author(s):

F Widmer ◽

P Caroni

Keyword(s):

Phosphorylation Site ◽

Growth Cones ◽

Cell Cortex ◽

Membrane Association ◽

Major Protein ◽

Cell Periphery ◽

Cortical Actin ◽

Cortical Cytoskeleton

The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of axonal growth cones, developing nerve terminals, regenerating axons, and adult central nervous system areas associated with plasticity. It is a cytosolic protein associated with the cortical cytoskeleton and the plasmalemma. Membrane association of GAP-43 is mediated by palmitoylation at Cys3Cys4. In vitro and in vivo, phosphorylation by PKC exclusively involves Ser41 of mammalian GAP-43 (corresponding to Ser42 in the chick protein). To identify aspects of GAP-43 function, we analyzed the actions of wild-type, membrane-association, and phosphorylation-site mutants of GAP-43 in nonneuronal cell lines. The GAP-43 constructs were introduced in L6 and COS-7 cells by transient transfection. Like the endogenous protein in neurons and their growth cones, GAP-43 in nonneuronal cells associated with the cell periphery. GAP-43 accumulated in the pseudopods of spreading cells and appeared to interact with cortical actin-containing filaments. Spreading L6 cells expressing high levels of recombinant protein displayed a characteristic F-actin labeling pattern consisting of prominent radial arrays of peripheral actin filaments. GAP-43 had dramatic effects on local surface morphology. Characteristic features of GAP-43-expressing cells were irregular cell outlines with prominent and numerous filopodia. The effects of GAP-43 on cell morphology required association with the cell membrane, since GAP-43(Ala3Ala4), a mutant that failed to associate with the cell cortex, had no morphogenetic activity. Two GAP-43 phosphorylation mutants (Ser42 to Ala42 preventing and Ser42 to Asp42 mimicking phosphorylation by PKC) modulated the effects of GAP-43 in opposite ways. Cells expressing GAP-43(Asp42) spread extensively and displayed large and irregular membranous extensions with little filopodia, whereas GAP-43(Ala42) produced small, poorly spreading cells with numerous short filopodia. Therefore, GAP-43 influences cell surface behavior and phosphorylation modulates its activity. The presence of GAP-43 in growing axons and developing nerve termini may affect the behavior of their actin-containing cortical cytoskeleton in a regulatable manner.

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The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro

The Journal of Cell Biology ◽

10.1083/jcb.200106089 ◽

2001 ◽

Vol 154 (6) ◽

pp. 1209-1224 ◽

Cited By ~ 178

Author(s):

Åsa E.Y. Engqvist-Goldstein ◽

Robin A. Warren ◽

Michael M. Kessels ◽

James H. Keen ◽

John Heuser ◽

...

Keyword(s):

Binding Protein ◽

Coiled Coil ◽

Actin Binding ◽

Live Cells ◽

Cell Cortex ◽

Coated Pits ◽

Actin Binding Protein ◽

Real Time Analysis

Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R–YFP and DsRed–clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of ‘unroofed’ cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1R's properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids.

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