ABSTRACT
Spatially resolved omics technologies reveal context-dependent cellular regulatory networks in tissues of interest. Beyond transcriptome analysis, information on epigenetic traits and chromatin accessibility can provide further insights on gene regulation in health and disease. Nevertheless, compared to the enormous advancements in spatial transcriptomics technologies, the field of spatial epigenomics is much younger and still underexplored. In this study, we report laser capture microdissection coupled to ATAC-seq (LCM-ATAC-seq) applied to fresh frozen samples for the spatial characterization of chromatin accessibility. We first demonstrate the efficient use of LCM coupled to in situ tagmentation and evaluate its performance as a function of cell number, microdissected areas, and tissue type. Further, we demonstrate its use for the targeted chromatin accessibility analysis of discrete contiguous or scattered cell populations in tissues via single-nuclei capture based on immunostaining for specific cellular markers.
Subject(s)
Chromatin Immunoprecipitation Sequencing , Chromatin , Chromatin/genetics , Laser Capture Microdissection , Gene Expression Profiling , FreezingABSTRACT
Neuropilin 1 (Nrp1) is a coreceptor for vascular endothelial growth factor A165 (VEGF-A165, VEGF-A164 in mice) and semaphorin 3A (SEMA3A). Nevertheless, Nrp1 null embryos display vascular defects that differ from those of mice lacking either VEGF-A164 or Sema3A proteins. Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Taken together, these data suggest that the vascular phenotype caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in ECs, such as adhesion to the extracellular matrix. By using RNA interference and rescue with wild-type and mutant constructs, we show here that Nrp1 through its cytoplasmic SEA motif and independently of VEGF-A165 and SEMA3A specifically promotes alpha5beta1-integrin-mediated EC adhesion to fibronectin that is crucial for vascular development. We provide evidence that Nrp1, while not directly mediating cell spreading on fibronectin, interacts with alpha5beta1 at adhesion sites. Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active alpha5beta1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with alpha5beta1, and the associated motor myosin VI (Myo6) support active alpha5beta1 endocytosis and EC adhesion to fibronectin. In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active alpha5beta1. Nrp1 modulation of alpha5beta1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice.
Subject(s)
Carrier Proteins/metabolism , Endothelium, Vascular/metabolism , Integrin alpha5beta1/metabolism , Neuropeptides/metabolism , Neuropilin-1/metabolism , Signal Transduction , Adaptor Proteins, Signal Transducing , Animals , Carrier Proteins/genetics , Cell Adhesion , Endothelium, Vascular/cytology , Fibronectins/genetics , Fibronectins/metabolism , Humans , Integrin alpha5beta1/genetics , Mice , Mice, Knockout , Neovascularization, Physiologic , Neuropeptides/genetics , Neuropilin-1/antagonists & inhibitors , Neuropilin-1/genetics , Protein Binding , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Umbilical Arteries/cytology , Umbilical Arteries/metabolismABSTRACT
We have used fluorescence lifetime imaging (FLIM) to study actin and plasma membrane dynamics in B16-F1 melanoma cells. In the absence of a FRET acceptor, significant changes in the fluorescence lifetime of GFP were induced simply by linking the fluorophore to different functional probes, including beta-actin, the PH domains of PLCdelta and Akt, the Ras farnesylation signal, and the neuromodulin palmitoylation signal (MEM). In contrast, the lifetime of GFP-actin was constant despite the many different local environments of G- and F-actin within the cell. Treatment with cytochalasin D but not latrunculin A significantly shortened the lifetime of GFP-beta-actin in the absence of a FRET acceptor. Robust lifetime shifts were observed using either a GFP-RFP chimera or co-transfection of GFP-MEM with RFP-MEM. In contrast to previous reports we observed a photobleaching-dependent change in the lifetime of GFP which could complicate the interpretation of FRET experiments. Of the membrane probes tested only the fluorescence lifetime of GFP-Akt was influenced by the presence of mRFP-actin, suggesting that the cortical actin meshwork is associated with a PIP3-enriched compartment of the plasma membrane. These results will aid in the design of new FRET-based approaches to study cytoskeletal interactions at the molecular level.
Subject(s)
Actin Cytoskeleton/ultrastructure , Cell Membrane/ultrastructure , Fluorescence Resonance Energy Transfer/methods , Microscopy, Fluorescence , Actin Cytoskeleton/metabolism , Animals , Cell Membrane/chemistry , Cell Membrane/metabolism , Green Fluorescent Proteins/metabolism , Membrane Microdomains/metabolism , Membrane Microdomains/ultrastructure , Mice , Phosphatidylinositol Phosphates/analysisABSTRACT
Total internal reflection fluorescence microscopy (TIRF-M) has become an increasingly popular tool to study events in close proximity to the cell cortex, such as cell adhesion (Axelrod, J Cell Biol 89:141-145, 1981; Gingell et al., J Cell Biol 100:1334-1338, 1985; Patel et al., J Cell Sci 121:1159-1164, 2008), actin (Bretschneider et al., Curr Biol 14:1-10, 2004; Gerisch, Biophys J 87:3493-3503, 2004; Merrifield et al., Nat Cell Biol 4:691-698, 2002), and membrane dynamics (Oheim et al., Eur Biophys J 27:83-98, 1998; Steyer et al., Nature 388:474-478, 1997; Weisswange et al., J Cell Sci 118:4375-4380, 2005). In TIRF-M, dim fluorescence from cortical structures can be imaged with high contrast despite large cytoplasmic background from the bulk of the cell body. With any imaging method, standard samples are required to ensure correct alignment and monitor system performance over time. Here, we describe procedures for the production and use of a test sample to characterise and optimize TIRF system performance.
Subject(s)
Microscopy, Fluorescence/methods , Algorithms , Calibration , Fluorescein/chemistry , Microscopy, Fluorescence/instrumentation , Microscopy, Fluorescence/standards , Microspheres , Reference Standards , SoftwareABSTRACT
Fascin is an actin-bundling protein involved in filopodia assembly and cancer invasion and metastasis of multiple epithelial cancer types. Fascin forms stable actin bundles with slow dissociation kinetics in vitro and is regulated by phosphorylation of serine 39 by protein kinase C (PKC). Cancer cells use invasive finger-like protrusions termed invadopodia to invade into and degrade extracellular matrix. Invadopodia have highly dynamic actin that is assembled by both Arp2/3 complex and formins; they also contain components of membrane trafficking machinery such as dynamin and cortactin and have been compared with focal adhesions and podosomes. We show that fascin is an integral component of invadopodia and that it is important for the stability of actin in invadopodia. The phosphorylation state of fascin at S39, a PKC site, contributes to its regulation at invadopodia. We further implicate fascin in invasive migration into collagen I-Matrigel gels and particularly in cell types that use an elongated mesenchymal type of motility in 3D. We provide a potential molecular mechanism for how fascin increases the invasiveness of cancer cells, and we compare invadopodia with invasive filopod-like structures in 3D.
Subject(s)
Actins/metabolism , Carrier Proteins/metabolism , Cell Membrane Structures/metabolism , Microfilament Proteins/metabolism , Neoplasm Invasiveness/physiopathology , Blotting, Western , Carboxylic Acids , Cell Line, Tumor , Collagen , Drug Combinations , Fluorescence Recovery After Photobleaching , Humans , Laminin , Microscopy, Fluorescence , Models, Biological , Phosphorylation , Protein Kinase C/metabolism , Proteoglycans , RNA, Small Interfering/geneticsABSTRACT
LIM kinases 1 and 2 (LIMK1/2) are centrally positioned regulators of actin cytoskeleton dynamics. Using siRNA-mediated knockdown or a novel small molecule inhibitor, we show LIMK is required for path generation by leading tumor cells and nontumor stromal cells during collective tumor cell invasion. LIMK inhibition lowers cofilin phosphorylation, F-actin levels, serum response factor transcriptional activity and collagen contraction, and reduces invasion in three-dimensional invasion assays. Although motility was unaffected, LIMK inhibition impairs matrix protein degradation and invadopodia formation associated with significantly faster recovery times in FRAP assays indicative of reduced F-actin stability. When LIMK is knocked down in MDA-MB-231 cells, they lose the ability to lead strands of collectively invading cells. Similarly, when LIMK activity is blocked in cancer-associated fibroblasts, they are unable to lead the collective invasion of squamous carcinoma cells in an organotypic skin model. These results show that LIMK is required for matrix remodeling activities for path generation by leading cells in collective invasion.
Subject(s)
Lim Kinases/physiology , Neoplasm Invasiveness , Stromal Cells/enzymology , Actin Depolymerizing Factors/metabolism , Actins/metabolism , Cell Line, Tumor , Extracellular Matrix/metabolism , Humans , Lim Kinases/antagonists & inhibitors , Phosphorylation , Protein Stability , RNA InterferenceABSTRACT
As well as providing a structural framework, the actin cytoskeleton plays integral roles in cell death, survival, and proliferation. The disruption of the actin cytoskeleton results in the activation of the c-Jun N-terminal kinase (JNK) stress-activated protein kinase (SAPK) pathway; however, the sensor of actin integrity that couples to the JNK pathway has not been characterized in mammalian cells. We now report that the mammalian Ste20-like (MST) kinases mediate the activation of the JNK pathway in response to the disruption of the actin cytoskeleton. One consequence of actin disruption is the JNK-mediated stabilization of p21(Waf1/Cip1) (p21) via the phosphorylation of Thr57. The expression of MST1 or MST2 was sufficient to stabilize p21 in a JNK- and Thr57-dependent manner, while the stabilization of p21 by actin disruption required MST activity. These data indicate that, in addition to being components of the Salvador-Warts-Hippo tumor suppressor network and binding partners of c-Raf and the RASSF1A tumor suppressor, MST kinases serve to monitor cytoskeletal integrity and couple via the JNK SAPK pathway to the regulation of a key cell cycle regulatory protein.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cytoskeleton/metabolism , Mitogen-Activated Protein Kinase 8/metabolism , Protein Serine-Threonine Kinases/metabolism , Actins/metabolism , Animals , Cyclin-Dependent Kinase Inhibitor p21/genetics , Emetine/metabolism , Enzyme Activation , Enzyme Stability , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Mice , Mitogen-Activated Protein Kinase 8/genetics , Mutagenesis, Site-Directed , NIH 3T3 Cells , Protein Serine-Threonine Kinases/genetics , Protein Synthesis Inhibitors/metabolism , RNA Interference , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Serine-Threonine Kinase 3 , Signal Transduction/physiologyABSTRACT
FERM domain proteins, including talins, ERMs, FAK and certain myosins, regulate connections between the plasma membrane, cytoskeleton and extracellular matrix. Here we show that FrmA, a Dictyostelium discoideum protein containing two talin-like FERM domains, plays a major role in normal cell shape, cell-substrate adhesion and actin cytoskeleton organisation. Using total internal reflection fluorescence (TIRF) microscopy we show that FrmA-null cells are more adherent to substrate than wild-type cells because of an increased number, persistence and mislocalisation of paxillin-rich cell-substrate adhesions, which is associated with decreased motility. We show for the first time that talinA colocalises with paxillin at the distal ends of filopodia to form cell-substrate adhesions and indeed arrives prior to paxillin. After a period of colocalisation, talin leaves the adhesion site followed by paxillin. Whereas talinA-rich spots turnover prior to the arrival of the main body of the cell, paxillin-rich spots turn over as the main body of the cell passes over it. In FrmA-null cells talinA initially localises to cell-substrate adhesion sites at the distal ends of filopodia but paxillin is instead localised to stabilised adhesion sites at the periphery of the main cell body. This suggests a model for cell-substrate adhesion in Dictyostelium whereby the talin-like FERM domains of FrmA regulate the temporal and spatial control of talinA and paxillin at cell-substrate adhesion sites, which in turn controls adhesion and motility.