Transcriptional Dysregulation and Impaired Neuronal Activity in FMR1 Knock-Out and Fragile X Patients' iPSC-Derived Models.

Fragile X syndrome (FXS) is caused by a repression of the FMR1 gene that codes the Fragile X mental retardation protein (FMRP), an RNA binding protein involved in processes that are crucial for proper brain development. To better understand the consequences of the absence of FMRP, we analyzed gene expression profiles and activities of cortical neural progenitor cells (NPCs) and neurons obtained from FXS patients' induced pluripotent stem cells (IPSCs) and IPSC-derived cells from FMR1 knock-out engineered using CRISPR-CAS9 technology. Multielectrode array recordings revealed in FMR1 KO and FXS patient cells, decreased mean firing rates; activities blocked by tetrodotoxin application. Increased expression of presynaptic mRNA and transcription factors involved in the forebrain specification and decreased levels of mRNA coding AMPA and NMDA subunits were observed using RNA sequencing on FMR1 KO neurons and validated using quantitative PCR in both models. Intriguingly, 40% of the differentially expressed genes were commonly deregulated between NPCs and differentiating neurons with significant enrichments in FMRP targets and autism-related genes found amongst downregulated genes. Our findings suggest that the absence of FMRP affects transcriptional profiles since the NPC stage, and leads to impaired activity and neuronal differentiation over time, which illustrates the critical role of FMRP protein in neuronal development.

CRISPR-clear imaging of melanin-rich B16-derived solid tumors.

Tissue clearing combined with deep imaging has emerged as a powerful technology to expand classical histological techniques. Current techniques have been optimized for imaging sparsely pigmented organs such as the mammalian brain. In contrast, melanin-rich pigmented tissue, of great interest in the investigation of melanomas, remains challenging. To address this challenge, we have developed a CRISPR-based gene editing approach that is easily incorporated into existing tissue-clearing workflows such the PACT clearing method. We term this method CRISPR-Clear. We demonstrate its applicability to highly melanin-rich B16-derived solid tumors, including one made transgenic for HER2, constituting one of very few syngeneic mouse tumors that can be used in immunocompetent models. We demonstrate the utility in detailed tumor characterization by staining for targeting antibodies and nanoparticles, as well as expressed fluorescent proteins. With CRISPR-Clear we have unprecedented access to optical interrogation in considerable portions of intact melanoma tissue for stained surface markers, expressed fluorescent proteins, of subcellular compartments, and of the vasculature.

Magnetically Single-Cell Virus Stamping.

Single-cell engineering via virus based genetic manipulation allows the possibility of understanding of complex tissues. However, current delivery methods for the genetic engineering of single cells via viral transduction suffer from limitations that restrict their application. Here I present a protocol describing a precise technique which can be used for the targeted virus infection of single cells in a monolayer of cells that is optically accessible. The protocol, demonstrated here by stamping cultured Hela cells with lentiviruses (LVs), completes in a few minutes and allows stable transgene expression within a few days, at success rates approaching 80%.

The SHREAD gene therapy platform for paracrine delivery improves tumor localization and intratumoral effects of a clinical antibody.

The goal of cancer-drug delivery is to achieve high levels of therapeutics within tumors with minimal systemic exposure that could cause toxicity. Producing biologics directly in situ where they diffuse and act locally is an attractive alternative to direct administration of recombinant therapeutics, as secretion by the tumor itself provides high local concentrations that act in a paracrine fashion continuously over an extended duration (paracrine delivery). We have engineered a SHielded, REtargeted ADenovirus (SHREAD) gene therapy platform that targets specific cells based on chosen surface markers and converts them into biofactories secreting therapeutics. In a proof of concept, a clinically approved antibody is delivered to orthotopic tumors in a model system in which precise biodistribution can be determined using tissue clearing with passive CLARITY technique (PACT) with high-resolution three-dimensional imaging and feature quantification within the tumors made transparent. We demonstrate high levels of tumor cell-specific transduction and significant and durable antibody production. PACT gives a localized quantification of the secreted therapeutic and allows us to directly observe enhanced pore formation in the tumor and destruction of the intact vasculature. In situ production of the antibody led to an 1,800-fold enhanced tumor-to-serum antibody concentration ratio compared to direct administration. Our detailed biochemical and microscopic analyses thus show that paracrine delivery with SHREAD could enable the use of highly potent therapeutic combinations, including those with systemic toxicity, to reach adequate therapeutic windows.

Kin discrimination in social yeast is mediated by cell surface receptors of the Flo11 adhesin family.

Microorganisms have evolved specific cell surface molecules that enable discrimination between cells from the same and from a different kind. Here, we investigate the role of Flo11-type cell surface adhesins from social yeasts in kin discrimination. We measure the adhesion forces mediated by Flo11A-type domains using single-cell force spectroscopy, quantify Flo11A-based cell aggregation in populations and determine the Flo11A-dependent segregation of competing yeast strains in biofilms. We find that Flo11A domains from diverse yeast species confer remarkably strong adhesion forces by establishing homotypic interactions between single cells, leading to efficient cell aggregation and biofilm formation in homogenous populations. Heterotypic interactions between Flo11A domains from different yeast species or Saccharomyces cerevisiae strains confer weak adhesive forces and lead to efficient strain segregation in heterogenous populations, indicating that in social yeasts Flo11A-mediated cell adhesion is a major mechanism for kin discrimination at species and sub-species levels. These findings, together with our structure and mutation analysis of selected Flo11A domains, provide a rationale of how cell surface receptors have evolved in microorganisms to mediate kin discrimination.

Magnetically guided virus stamping for the targeted infection of single cells or groups of cells.

To understand and control complex tissues, the ability to genetically manipulate single cells is required. However, current delivery methods for the genetic engineering of single cells, including viral transduction, suffer from limitations that restrict their application. Here we present a protocol that describes a versatile technique that can be used for the targeted viral infection of single cells or small groups of cells in any tissue that is optically accessible. First, cells of interest are selected using optical microscopy. Second, a micropipette-loaded with magnetic nanoparticles to which viral particles are bound-is brought into proximity of the cell of interest, and a magnetic field is applied to guide the viral nanoparticles into cellular contact, leading to transduction. The protocol, exemplified here by stamping cultured neurons with adeno-associated viruses (AAVs), is completed in a few minutes and allows stable transgene expression within a few days, at success rates that approach 80%. We outline how this strategy is applied to single-cell infection in complex tissues, and is feasible both in organoids and in vivo.

Virus stamping for targeted single-cell infection in vitro and in vivo.

Genetic engineering by viral infection of single cells is useful to study complex systems such as the brain. However, available methods for infecting single cells have drawbacks that limit their applications. Here we describe 'virus stamping', in which viruses are reversibly bound to a delivery vehicle-a functionalized glass pipette tip or magnetic nanoparticles in a pipette-that is brought into physical contact with the target cell on a surface or in tissue, using mechanical or magnetic forces. Different single cells in the same tissue can be infected with different viruses and an individual cell can be simultaneously infected with different viruses. We use rabies, lenti, herpes simplex, and adeno-associated viruses to drive expression of fluorescent markers or a calcium indicator in target cells in cell culture, mouse retina, human brain organoid, and the brains of live mice. Virus stamping provides a versatile solution for targeted single-cell infection of diverse cell types, both in vitro and in vivo.

Nanomechanical mapping of first binding steps of a virus to animal cells.

Viral infection is initiated when a virus binds to cell surface receptors. Because the cell membrane is dynamic and heterogeneous, imaging living cells and simultaneously quantifying the first viral binding events is difficult. Here, we show an atomic force and confocal microscopy set-up that allows the surface receptor landscape of cells to be imaged and the virus binding events within the first millisecond of contact with the cell to be mapped at high resolution (<50 nm). We present theoretical approaches to contour the free-energy landscape of early binding events between an engineered virus and cell surface receptors. We find that the first bond formed between the viral glycoprotein and its cognate cell surface receptor has relatively low lifetime and free energy, but this increases as additional bonds form rapidly (≤1 ms). The formation of additional bonds occurs with positive allosteric modulation and the three binding sites of the viral glycoprotein are quickly occupied. Our quantitative approach can be readily applied to study the binding of other viruses to animal cells.

The biomechanical properties of an epithelial tissue determine the location of its vasculature.

An important question is how growing tissues establish a blood vessel network. Here we study vascular network formation in pancreatic islets, endocrine tissues derived from pancreatic epithelium. We find that depletion of integrin-linked kinase (ILK) in the pancreatic epithelial cells of mice results in glucose intolerance due to a loss of the intra-islet vasculature. In turn, blood vessels accumulate at the islet periphery. Neither alterations in endothelial cell proliferation, apoptosis, morphology, Vegfa expression and VEGF-A secretion nor 'empty sleeves' of vascular basement membrane are found. Instead, biophysical experiments reveal that the biomechanical properties of pancreatic islet cells, such as their actomyosin-mediated cortex tension and adhesive forces to endothelial cells, are significantly changed. These results suggest that a sorting event is driving the segregation of endothelial and epithelial cells and indicate that the epithelial biomechanical properties determine whether the blood vasculature invades or envelops a growing epithelial tissue.

Assay for characterizing the recovery of vertebrate cells for adhesion measurements by single-cell force spectroscopy.

Single-cell force spectroscopy (SCFS) is becoming a widely used method to quantify the adhesion of a living cell to a substrate, another cell or tissue. The high sensitivity of SCFS permits determining the contributions of individual cell adhesion molecules (CAMs) to the adhesion force of an entire cell. However, to prepare adherent cells for SCFS, they must first be detached from tissue-culture flasks or plates. EDTA and trypsin are often applied for this purpose. Because cellular properties can be affected by this treatment, cells need to recover before being further characterized by SCFS. Here we introduce atomic force microscopy (AFM)-based SCFS to measure the mechanical and adhesive properties of HeLa cells and mouse embryonic kidney fibroblasts while they are recovering after detachment from tissue-culture. We find that mechanical and adhesive properties of both cell lines recover quickly (<10 min) after detachment using EDTA, while trypsin-detached fibroblasts require >60 min to fully recover. Our assay introduced to characterize the recovery of mammalian cells after detachment can in future be used to estimate the recovery behavior of other adherent cell types.

Deciphering teneurin domains that facilitate cellular recognition, cell-cell adhesion, and neurite outgrowth using atomic force microscopy-based single-cell force spectroscopy.

Teneurins are evolutionarily conserved transmembrane receptors that function as axon guidance and target selection molecules in the developing nervous system. How teneurins recognize each other, whether they establish neuronal adhesion, and which teneurin specific interactions guide neurons remains to be determined. To reveal insight into these pertinent questions we combine atomic force microscopy-based single-cell force spectroscopy with genetic engineering and quantify the interactions teneurins establish between animal cells. Using a combinatorial approach of deletions and swaps of teneurin-1 and teneurin-2 domains, we unravel that teneurins use their NHL (NCL-1, HT2A, and Lin-41) domain to select homophilic teneurins from adjacent cells. This homophilic recognition of teneurins initiates cell-cell adhesion that, dependent on the intracellular domain, strengthens over time. Neurite outgrowth assays show that establishing and strengthening of teneurin-mediated homophilic cell-cell adhesion is required to stop outgrowth. On the basis of the results, we introduce a molecular model of teneurin domains that specify cellular recognition, adhesion strengthening, and neuronal pathfinding. The combined force spectroscopy and genetic approach can be applied to quantitatively decipher the contribution of any neuronal receptor domain and more generally of a given cell surface receptor domain to cell-cell recognition and adhesion.

A practical guide to quantify cell adhesion using single-cell force spectroscopy.

Quantitative analysis of cellular interactions with the extracellular environment is necessary to gain an understanding of how cells regulate adhesion in the development and maintenance of multicellular organisms, and how changes in cell adhesion contribute to diseases. We provide a practical guide to quantify the adhesive strength of living animal cells to various substrates using atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS). We describe how to control cell state and attachment to the AFM cantilever, how to functionalize supports for SCFS measurements, how to conduct cell adhesion measurements, and how to analyze and interpret the recorded SCFS data. This guide is intended to assist newcomers in the field to perform AFM-based SCFS measurements.