Towards Synthetic Life: Establishing a Minimal Segrosome for the Rational Design of Biomimetic Systems

DNA segregation is a fundamental life process, crucial for renewal, reproduction and propagation of all forms of life. Hence, a dedicated segregation machinery, a segrosome, must function reliably also in the context of a minimal cell. Conceptionally, the development of such a minimal cell follows a minimalistic approach, aiming at engineering a synthetic entity only consisting of the essential key elements necessary for a cell to survive. In this thesis, various prokaryotic segregation systems were explored as possible candidates for a minimal segrosome. Such a minimal segrosome could be applied for the rational design of biomimetic systems including, but not limited to, a minimal cell. DNA segregation systems of type I (ParABS) and type II (ParMRC) were compared for ensuring genetic stabilities in vivo using vectors derived from the natural secondary chromosome of Vibrio cholerae. The type II segregation system R1-ParMRC was chosen as the most promising candidate for a minimal segrosome, and it was characterized and reconstituted in vitro. This segregation system was encapsulated into biomimetic micro-compartments and its lifetime prolonged by coupling to ATP-regenerating as well as oxygen-scavenging systems. The segregation process was coupled to in vitro DNA replication using DNA nanoparticles as a mimic of the condensed state of chromosomes. Furthermore, another type II segregation system originating from the pLS20 plasmid from Bacillus subtilis (Alp7ARC) was reconstituted in vitro as a secondary orthogonal segrosome. Finally, a chimeric RNA segregation system was engineered that could be applied for an RNA-based protocell. Overall, this work demonstrates successful bottom-up assemblies of functional molecular machines that could find applications in biomimetic systems and lead to a deeper understanding of living systems

Production and analysis of synthetic Cascade variants

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR assoziiert) ist ein adaptives Immunsystem in Archaeen und Bakterien, das fremdes genetisches Material mit Hilfe von Ribonukleoprotein-Komplexen erkennt und zerstört. Diese Komplexe bestehen aus einer CRISPR RNA (crRNA) und Cas Proteinen. CRISPR-Cas Systeme sind in zwei Hauptklassen und mehrere Typen unterteilt, abhängig von den beteiligten Cas Proteinen. In Typ I Systemen sucht ein Komplex namens Cascade (CRISPR associated complex for antiviral defence) nach eingedrungener viraler DNA während einer Folgeinfektion und bindet die zu der eingebauten crRNA komplementäre Sequenz. Anschließend wird die Nuklease/Helikase Cas3 rekrutiert, welche die virale DNA degradiert (Interferenz). Das Typ I System wird in mehrere Subtypen unterteilt, die Unterschiede im Aufbau von Cascade vorweisen. Im Fokus dieser Arbeit steht eine minimale Cascade-Variante aus Shewanella putrefaciens CN-32. Im Vergleich zur gut untersuchten Typ I-E Cascade aus Escherichia coli fehlen in diesem Komplex zwei Untereinheiten, die gewöhnlicher Weise für die Zielerkennung benötigt werden. Dennoch ist der Komplex aktiv. Rekombinante I-Fv Cascade wurde bereits aus E. coli aufgereinigt und es war möglich, den Komplex zu modifizieren, indem das Rückgrat entweder verlängert oder verkürzt wurde. Dadurch wurden synthetische Varianten mit veränderter Protein-Stöchiometrie erzeugt. In der vorliegenden Arbeit wurde I-Fv Cascade weiter mit in vitro Methoden untersucht. So wurde die Bindung von Ziel-DNA beobachtet und die 3D Struktur zeigt, dass strukturelle Veränderungen im Komplex die fehlenden Untereinheiten ersetzen, möglicherweise um viralen Anti-CRISPR Proteinen zu entgehen. Die Nuklease/Helikase dieses Systems, Cas2/3fv, ist eine Fusion des Cas3 Proteins mit dem Interferenz-unabhängigen Protein Cas2. Ein unabhängiges Cas3fv ohne Cas2 Untereinheit wurde aufgereinigt und in vitro Assays zeigten, dass dieses Protein sowohl freie ssDNA als auch Cascadegebundene Substrate degradiert. Das komplette Cas2/3fv Protein bildet einen Komplex mit dem Protein Cas1 und zeigt eine reduzierte Aktivität gegenüber freier ssDNA, möglicherweise als Regulationsmechanismus zur Vermeidung von unspezifischer Aktivität. Weiterhin wurde ein Prozess namens „RNA wrapping“ etabliert. Synthetische Cascade-Komplexe wurden erzeugt, in denen die grundlegende RNA-Bindung des charakteristischen Cas7fv RückgratProteins auf eine ausgewählte RNA gelenkt wird. Diese spezifische Komplexbildung kann in vivo durch eine Repeat-Sequenz der crRNA stromaufwärts der Zielsequenz und durch Bindung des Cas5fv Proteins initiiert werden. Die erzeugten Komplexe beinhalten die ersten 100 nt der markierten RNA, die anschließend isoliert werden kann. Innerhalb der Komplexe ist die RNA stabilisiert und geschützt vor Degradation durch RNasen. Komplexbildung kann außerdem genutzt werden, um ReportergenTranskripte stillzulegen. Zusätzlich wurden erste Hinweise geliefert, dass das Rückgrat der synthetischen Komplexe durch Fusion mit weiteren Reporterproteinen modifiziert werden kann.CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) is an adaptive immune system of Archaea and Bacteria. It is able to target and destroy foreign genetic material with ribonucleoprotein complexes consisting of CRISPR RNAs (crRNAs) and certain Cas proteins. CRISPR-Cas systems are classified in two major classes and multiple types, according to the involved Cas proteins. In type I systems, a ribonucleoprotein complex called Cascade (CRISPR associated complex for antiviral defence) scans for invading viral DNA during a recurring infection and binds the sequence complementary to the incorporated crRNA. After target recognition, the nuclease/helicase Cas3 is recruited and subsequently destroys the viral DNA in a step termed interfere nce. Multiple subtypes of type I exist that show differences in the Cascade composition. This work focuses on a minimal Cascade variant found in Shewanella putrefaciens CN-32. In comparison to the well-studied type I-E Cascade from Escherichia coli, this complex is missing two proteins usually required for target recognition, yet it is still able to provide immunity. Recombinant I-Fv Cascade was previously purified from E. coli and it was possible to modulate the complex by extending or shortening the backbone, resulting in synthetic variants with altered protein stoichiometry. In the present study, I-Fv Cascade was further analyzed by in vitro methods. Target binding was observed and the 3D structure revealed structural variations that replace the missing subunits, potentially to evade viral anti-CRISPR proteins. The nuclease/helicase of this system, Cas2/3fv, is a fusion of the Cas3 protein with the interference-unrelated protein Cas2. A standalone Cas3fv was purified without the Cas2 domain and in vitro cleavage assays showed that Cas3fv degrades both free ssDNA as well as Cascade-bound substrates. The complete Cas2/3fv protein forms a complex with the protein Cas1 and was shown to reduce cleave of free ssDNA, potentially as a regulatory mechanism against unspecific cleavage. Furthermore, we established a process termed “RNA wrapping”. Synthetic Cascade assemblies can be created by directing the general RNA-binding ability of the characteristic Cas7fv backbone protein on an RNA of choice such as reporter gene transcripts. Specific complex formation can be initiated in vivo by including a repeat sequence from the crRNA upstream a given target sequence and binding of the Cas5fv protein. The created complexes contain the initial 100 nt of the tagged RNA which can be isolated afterwards. While incorporated in complexes, RNA is stabilized and protected from degradation by RNases. Complex formation can be used to silence reporter gene transcripts. Furthermore, we provided initial indications that the backbone of synthetic complexes can be modified by addition of reporter proteins

What Makes a Muslim Intellectual? On the Pro's and Con's of a Category

<p style="margin-right: 1cm; margin-bottom: 0cm; line-height: 100%;" lang="en-GB">At its core, this essay contains a substantiated plea for bringing about conceptual clarity to the notion of “Muslim intellectual”, which the frequent and highly ideologically charged public usage of this term seems to distort. In search for a sound analytical concept of “intellectual” first, relevant sociological and philosophical deliberations are highlighted, indicating that both of their notions differ to such an extent that their applicability to academic pursuit must be doubted. Yet, by discussing some considerations by a Study of Islam open to the approaches of the Social Sciences a possible framework for an analytically meaningful concept of “Muslim intellectual” is presented. At the same time, however, arguments are presented for why those contemporary Muslim thinkers who are usually credited with being “Muslim intellectuals” would hardly fit the analytical criteria for such label.</p

Long-Term Results after DMEK (Descemet’s Membrane Endothelial Keratoplasty)

Ziel der Arbeit: Evaluation der langfristigen Ergebnisse sowie der Komplikationsrate nach Descemet’s Membran Endothelialen Keratoplastik (DMEK) Methoden: Eine cross-sectional, Fall-Serien Studie. Insgesamt wurden 230 Augen von 142 Patienten, die zwischen 2010 und 2014 eine DMEK an der Universitäts-Augenklinik Marburg bekommen haben, untersucht. Die best-korrigierte Sehschärfe (BCVA), die Refraktion, die zentrale Hornhautdicke, das Hornhautvolumen sowie die Endothelialzelldichte wurden als Parameter herangezogen und mit den präoperativen Befunden verglichen. Die Transplantat-Überlebensrate sowie die postoperativen Komplikationen wurden ebenfalls betrachtet. Ergebnisse: Die Nachbeobachtungszeit betrug 47 ± 13.3 Monate. Bei den Patienten die keine anderen okuläre Erkrankungen hatten hat sich die BCVA von 0.60 ± 0.32 logMAR präoperativ auf bis zu 0.10 ± 0.22 logMAR verbessert (201 Augen). 71.1% dieser Patienten hatten eine BCVA von 0.11 logMAR oder besser (≥ 0.8 dezimal), wobei 49.2% dieser Patienten eine volle BCVA von 0.00 logMAR oder besser erreicht haben. Die zentrale Hornhautdicke hat von 675 ± 112µm präoperativ auf 547 ± 52 µm in der letzten Follow-up Untersuchung abgenommen, und das Hornhautvolumen hat von 65.2 ± 8.4 mm2 präoperativ auf 61.9 ± 5.4 mm2 abgenommen. Der Endothelzellverlust lag bei 1392 ± 455 Zellen/mm², was einem durchschnittlichen Verlust von 54.7% der Transplantatzellen entspricht. Die Transplantat-Überlebensrate lag bei 92% mit einer durchschnittlichen Überlebenszeit von 76.6 ± 1.3 Monaten. Schlussfolgerung: DMEK bietet hohe visuelle Ergebnisse und sehr gute klinische Befunde, die mehrere Jahre nach der Operation stabil bleiben können. Durch die hohe Transplantat-Überlebensrate und die niedrige postoperative Komplikationsrate wird DMEK derzeit als erste Wahl zur Behandlung von Endothelzellerkrankungen eingesetzt

Establishment of surface functionalization methods for spore-based biosensors and implementation into sensor technologies for aseptic food processing

Aseptic processing has become a popular technology to increase the shelf-life of packaged products and to provide non-contaminated goods to the consumers. In 2017, the global aseptic market was evaluated to be about 39.5 billion USD. Many liquid food products, like juice or milk, are delivered to customers every day by employing aseptic filling machines. They can operate around 12,000 ready-packaged products per hour (e.g., Pure-Pak® Aseptic Filling Line E-PS120A). However, they need to be routinely validated to guarantee contamination-free goods. The state-of-the-art methods to validate such machines are by means of microbiological analyses, where bacterial spores are used as test organisms because of their high resistance against several sterilants (e.g., gaseous hydrogen peroxide). The main disadvantage of the aforementioned tests is time: it takes at least 36-48 hours to get the results, i.e., the products cannot be delivered to customers without the validation certificate. Just in this example, in 36 hours, 432,000 products would be on hold for dispatchment; if more machines are evaluated, this number would linearly grow and at the end, the costs (only for waiting for the results) would be considerably high. For this reason, it is very valuable to develop new sensor technologies to overcome this issue. Therefore, the main focus of this thesis is on the further development of a spore-based biosensor; this sensor can determine the viability of spores after being sterilized with hydrogen peroxide. However, the immobilization strategy as well as its implementation on sensing elements and a more detailed investigation regarding its operating principle are missing. In this thesis, an immobilization strategy is developed to withstand harsh conditions (high temperatures, oxidizing environment) for spore-based biosensors applied in aseptic processing. A systematic investigation of the surface functionalization’s effect (e.g., hydroxylation) on sensors (e.g., electrolyte-insulator semiconductor (EIS) chips) is presented. Later on, organosilanes are analyzed for the immobilization of bacterial spores on different sensor surfaces. The electrical properties of the immobilization layer are studied as well as its resistance to a sterilization process with gaseous hydrogen peroxide. In addition, a sensor array consisting of a calorimetric gas sensor and a spore-based biosensor to measure hydrogen peroxide concentrations and the spores’ viability at the same time is proposed to evaluate the efficacy of sterilization processes

Uniform convergence rates and uniform adaptive estimation in mixtures of regressions

In this thesis, we develop theoretical tools to examine estimators in non-parametric regression models in regard of uniform convergence rates and uniform adaptivity with respect to the smoothness of the parameter functions. Subsequently, those are applied to non-parametric regression models with Hölder-smooth parameter functions. One model is a mixture of Gaussian regressions and the other model is a mixture model with two components and an unspecified symmetric error distribution

Histone deacetylase 2-mediated deacetylation of the Ribonuclease 1 promoter in inflamed human endothelial cells

Endothelial cells (ECs) function as protective barrier to separate the blood from the surrounding tissue by conducting crucial roles in regulation and maintenance of vascular homeostasis, such as control of vessel permeability or coagulation. Therefore, dysfunction of the EC barrier due to inflammation, infection or injury can cause a variety of vascular pathologies, such as thrombosis or atherosclerosis. In this context, the circulating extracellular endonuclease Ribonuclease 1 (RNase1) was identified as a vessel- and tissue-protective enzyme and a potent regulator of vascular homeostasis. Upon acute inflammation, RNase1 functions as a natural counterpart to extracellular RNA (eRNA), a damage-associated molecular pattern, via degradation to protect the EC cell layer from excessive inflammation. However, long-term inflammation disrupts the RNase1-eRNA system. Thereby, eRNA accumulates in the extracellular space to induce massive proinflammatory cytokine release from circulating inflammatory cells, such as tumor necrosis factor alpha (TNF-α) or interleukin 1 beta (IL-1β). These cytokines negatively affect the EC layer by downregulation of RNase1 presumably through activation of histone deacetylases (HDACs). In this regard, this study investigated whether inflammation-mediated deacetylase function of HDACs suppresses RNase1 expression in human ECs through modulation of chromatin modifications. Proinflammatory stimulation with TNF-α or IL-1β of human umbilical vein endothelial cells significantly reduced RNase1 expression. Thus, identification of the RNASE1 promoter region and analysis of its chromatin state revealed the association of RNASE1 repression with deacetylation of histone 3 at lysine 27 and histone 4. The important role of HDACs in this process was further confirmed by administration of the specific class I HDAC1-3 inhibitor MS275 that successfully restored RNASE1 promoter acetylation and mRNA abundance upon TNF-α or IL-1β treatment. These results indicate an essential impact of HDAC1-3 in RNase1 regulation. Additionally, identification of specific HDACs involved in RNase1 regulation was obtained by chromatin immunoprecipitation kinetics confirming significant accumulation of HDAC2 at the RNASE1 promoter upon TNF-α stimulation. These findings were further validated by siRNA double knockdown of HDAC2 and its redundant enzyme HDAC1, which also recovered RNase1 mRNA abundance upon proinflammatory stimulation. In conclusion, our data identified HDAC2 as a crucial factor in RNase1 regulation in human ECs. HDAC2 is recruited to the RNASE1 promoter site to attenuate histone acetylation and suppress subsequent gene repression. This effect can be blocked by the specific HDAC inhibitor MS275 implicating the potential of HDAC inhibitors as novel therapeutic strategy to promote vascular integrity by preventing RNase1 downregulation in EC inflammation

Accurate Prediction of Core Properties for Chiral Molecules using Pseudo Potentials

Pseudo potentials (PPs) constitute perhaps the most common way to treat relativity, often in a formally non-relativistic framework, and reduce the electronic structure to the chemically relevant part. The drawback is that orbitals obtained in this picture (called pseudo orbitals (POs)) show a reduced nodal structure and altered amplitude in the vicinity of the nucleus, when compared to the corresponding molecular orbitals (MOs). Thus expectation values of operators localized in the spatial core region that are calculated with POs, deviate significantly from the same expectation values calculated with all-electron (AE) MOs. This study describes the reconstruction of AE MOs from POs, with a focus on POs generated by energy consistent pseudo Hamiltonians. The method reintroduces the nodal structure into the POs, thus providing an inexpensive and easily implementable method that allows to use nonrelativistic, efficiently calculated POs for good estimates of expectation values of core-like properties. The discussion of the method proceeds in two parts: Firstly, the reconstruction scheme is developed for atomic cases. Secondly, the scheme is discussed in the context of MO reconstruction and successfully applied to numerous numerical examples. Starting from the equations of the state-averaged multi-configuration self- consistent field method, used for the generation of energy consistent pseudo potentials, the electronic spectrum of the many-electron Hamiltonian is linked to the spectrum of the effective one-electron Fock operator by means of various models systems. This relation and the Topp–Hopfield–Kramers theorem, are used to show the shape-consistency of energy-consistent POs for atomic systems. Shape-consistency describes POs that follow distinct AOs exactly outside a core-radius r_core . In the cases presented here, shape-consistency holds to a high degree and it follows that in atomic systems every PO has one distinct partner in the set of AOs. The overlap integral between these two orbitals is close to one, as it is determined mainly by the spatial orbital parts outside r_core . Expanding, e.g., a 5s PO in occupied AOs, the 5s AOs will have the highest contribution. The POs itself contains contributions from high-energy unoccupied AOs as well (e.g. 15s), which damp the nodal structure of the POs near the nucleus. Consequently, neglecting contributions from unoccupied orbitals in a projection of the POs reintroduces the nodal structure. This approach is not directly suitable for the reconstruction of MOs, as they often need to be expanded in a full set of AOs at each atomic center, including all unoccupied orbitals, to properly account for the electron density distribution in the molecule. However, it is shown that the occupied MOs are well described by occupied and low-energy unoccupied AOs only and a mapping of the POs onto a basis containing only these orbitals reconstructs the nodal structure of the MO. The approach uses only standard integrals available in most quantum chemistry programs. The computational cost of these integrals scales with N^2 , where N is the number of basis functions. The most time consuming step is a Gram-Schmidt orthogonalization, which scales in this implementation with MN^2 , M being the number of reconstructed orbitals. The reconstruction method is subsequently tested: Valence orbitals of atomic, closed-shell systems were reconstructed numerically exactly. The influence of numerical parameters is investigated using the molecule BaF . It is shown that the method is basis set dependent: One has to ensure that the PO basis can be expanded exactly in the basis of AOs. Violating this rule of thumb may degrade the quality of reconstructed orbitals. Additionally, the representation of MOs by a linear combination of occupied and unoccupied AOs is investigated. For the exemplary systems, the shells included in the fitting procedure of the PP were sufficient. Reconstruction of the alkaline earth monofluorides showed that periodic trends can be reconstructed as well. Scaling of hyperfine structure parameters with increasing atomic number is discussed. For hydrogenic atoms, the scaling should be linear, whereas small deviations from the linear behavior were observed for molecules. The scaling laws computed from reconstructed and reference orbitals were almost identical. In this context, the failure of commonly used relativistic enhancement factors beyond atomic number 100 is discussed. Applicability of the method is also tested on parity violating properties for which the main contribution is generated by the valence orbitals near the nucleus. Symmetry-independence of the method is shown by successful reconstruction of orbitals of the tetrahedral PbCl_4 and chiral NWHClF. The reliable reconstruction of chemical trends is shown with the help of the NWHClF derivatives NWHBrF and NWHFI. The study of chiral compounds as, e.g., NWHClF and its group 17 derivatives, which have been proposed as paradigm for the detection of parity-violation in chiral molecules, remains of great importance. Especially the direct determination of absolute configuration of chiral centers is still non-trivial. The author contributed to this field with a self-written molecular dynamics (MD) program to simulate Coulomb explosions and thus to provide an insight especially into the early explosion stages directly after an instantaneous multi-ionization of the molecule CHBrClF, comparable to experiments using the Cold Target Recoil Ion Momentum Spectroscopy (COLTRIMS) technique. An algorithm for the determination of the investigated molecule’s absolute configuration from time-of-flight data and detection locations of molecular fragments is included in the program. The program was used to generate experiment-equivalent data which allowed for the first time the investigation of non-racemic mixtures by the analysis routines of the experiment. The MD program includes harmonic and anharmonic bond potentials. A charge-exchange model can model partial charges in early phases of the Coulomb explosion. Furthermore, Born–Oppenheimer MD simulations and statistical models are used to explain the relative abundance of products belonging to competing reaction channels, as obtained by photoion coincidence measurements. Additionally, qualitative statements about reaction branching ratios are made by comparing the partition functions of involved degrees of freedom. Analytic equations for partition functions of simple models are used to provide a simple formula allowing fast estimates of reaction branching ratios

Studies of Single-Molecule Dynamics in Microorganisms

Fluorescence microscopy is one of the most extensively used techniques in the life sciences. Considering the non-invasive sample preparation, enabling live-cell compliant imaging, and the specific fluorescence labeling, allowing for a specific visualization of virtually any cellular compound, it is possible to localize even a single molecule in living cells. This makes modern fluorescence microscopy a powerful toolbox. In the recent decades, the development of new, "super-resolution" fluorescence microscopy techniques, which surpass the diffraction limit, revolutionized the field. Single-Molecule Localization Microscopy (SMLM) is a class of super-resolution microscopy methods and it enables resolution of down to tens of nanometers. SMLM methods like Photoactivated Localization Microscopy (PALM), (direct) Stochastic Optical Reconstruction Microscopy ((d)STORM), Ground-State Depletion followed by Individual Molecule Return (GSDIM) and Point Accumulation for Imaging in Nanoscale Topography (PAINT) have allowed to investigate both, the intracellular spatial organization of proteins and to observe their real-time dynamics at the single-molecule level in live cells. The focus of this thesis was the development of novel tools and strategies for live-cell SingleParticle Tracking PALM (sptPALM) imaging and implementing them for biological research. In the first part of this thesis, I describe the development of new Photoconvertible Fluorescent Proteins (pcFPs) which are optimized for sptPALM lowering the phototoxic damage caused by the imaging procedure. Furthermore, we show that we can utilize them together with Photoactivatable Fluorescent Proteins (paFPs) to enable multi-target labeling and read-out in a single color channel, which significantly simplifies the sample preparation and imaging routines as well as data analysis of multi-color PALM imaging of live cells. In parallel to developing new fluorescent proteins, I developed a high throughput data analysis pipeline. I have implemented this pipeline in my second project, described in the second part of this thesis, where I have investigated the protein organization and dynamics of the CRISPR-Cas antiviral defense mechanism of bacteria in vivo at a high spatiotemporal level with the sptPALM approach. I was successful to show the differences in the target search dynamics of the CRISPR effector complexes as well as of single Cas proteins for different target complementarities. I have also first data describing longer-lasting bound-times between effector complex and their potential targets in vivo, for which only in vitro data has been available till today. In summary, this thesis is a significant contribution for both, the advances of current sptPALM imaging methods, as well as for the understanding of the native behavior of CRISPR-Cas systems in vivo

Transforming growth factor-beta targets Formin-like 2 for Angiopoietin-like 4 secretion during the epithelial mesenchymal transition

Epithelial to Mesenchymal transition (EMT) is a highly dynamic process that plays a crucial role in tumor progression and metastasis. While remodelling of the actin cytoskeleton is a hallmark of EMT, the responsible actin regulating factors are less well understood. Formins are involved in numerous cellular mechanisms, ranging from cytokinesis to cell adhesion and motility. The Rho-GTPase effectors of the formin family compromise the largest group of actin nucleators and are emerging as relevant pharmacological targets. A critical role of Formin-like 2 (FMNL2) in the assembly of junctional actin at newly forming cell-cell contacts in a 3D matrix has been described. This activity originates downstream of Rac1 and is in line with a physical association of FMNL2 and components of the cadherin-catenin complex. FMNL2 was further recently implicated in β1-integrin trafficking as a direct PKC target required for cancer cell invasion. Here we found that transforming growth factor-beta (TGFβ)-driven EMT leads to an upregulation of PKC resulting in the phosphorylation and activation of FMNL2 in epithelial cells. Proteomic screening for TGFβ-mediated phospho-FMNL2 binding partners identified the tumor promotor ANGPTL4 as a specific binding partner. ANGPTL4 has important roles in cancer development and progression including promoting invasion and metastasis. We found that FMNL2 and ANGPTL4 directly interact under TGF-induced EMT. Our data show that FMNL2 is a critical regulator of ANGPTL4 secretion. Secretion of ANGPTL4 is diminished upon loss of FMNL2 and its phosphorylation. We further observed that ANGPTL4 is sequestered in the Golgi apparatus colocalizing with markers of the trans-Golgi network. Live imaging of vesicle secretion from the Golgi confirmed the transient co-localization of ANGPTL4 and FMNL2. Moreover, ANGPTL4 and FMNL2 modulate cell-cell contact integrity and ANGPTL4 silenced cells fail to disassemble their underlying cell-cell contacts to execute EMT. This effect was further enhanced upon FMNL2 knockout using FMNL2 CRISPR/Cas9 cell line. However, re-introduction of ANGPTL4 restored the mesenchymal phenotype and prompted the dissolution of cell-cell adhesions. Finally, we found that cellular invasion promoted by TGFβ depends on FMNL2 and is reduced upon ANGPTL4 silencing. Taken together, our data point towards a crucial role of FMNL2 for EMT via ANGPTL4 secretion