Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development

De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). Here, we used Cul3 mouse models to evaluate the consequences of Cul3 mutations in vivo. Our results show that Cul3 haploinsufficient mice exhibit deficits in motor coordination as well as ASD-relevant social and cognitive impairments. Cul3 mutant brain displays cortical lamination abnormalities due to defective neuronal migration and reduced numbers of excitatory and inhibitory neurons. In line with the observed abnormal columnar organization, Cul3 haploinsufficiency is associated with decreased spontaneous excitatory and inhibitory activity in the cortex. At the molecular level, employing a quantitative proteomic approach, we show that Cul3 regulates cytoskeletal and adhesion protein abundance in mouse embryos. Abnormal regulation of cytoskeletal proteins in Cul3 mutant neuronal cells results in atypical organization of the actin mesh at the cell leading edge, likely causing the observed migration deficits. In contrast to these important functions early in development, Cul3 deficiency appears less relevant at adult stages. In fact, induction of Cul3 haploinsufficiency in adult mice does not result in the behavioral defects observed in constitutive Cul3 haploinsufficient animals. Taken together, our data indicate that Cul3 has a critical role in the regulation of cytoskeletal proteins and neuronal migration and that ASD-associated defects and behavioral abnormalities are primarily due to Cul3 functions at early developmental stages

Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development

De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 lead to autism spectrum disorder (ASD). In mouse, constitutive haploinsufficiency leads to motor coordination deficits as well as ASD-relevant social and cognitive impairments. However, induction of Cul3 haploinsufficiency later in life does not lead to ASD-relevant behaviors, pointing to an important role of Cul3 during a critical developmental window. Here we show that Cul3 is essential to regulate neuronal migration and, therefore, constitutive Cul3 heterozygous mutant mice display cortical lamination abnormalities. At the molecular level, we found that Cul3 controls neuronal migration by tightly regulating the amount of Plastin3 (Pls3), a previously unrecognized player of neural migration. Furthermore, we found that Pls3 cell-autonomously regulates cell migration by regulating actin cytoskeleton organization, and its levels are inversely proportional to neural migration speed. Finally, we provide evidence that cellular phenotypes associated with autism-linked gene haploinsufficiency can be rescued by transcriptional activation of the intact allele in vitro, offering a proof of concept for a potential therapeutic approach for ASDs

ISTA Thesis

Detachment of the cancer cells from the bulk of the tumor is the first step of metastasis, which is the primary cause of cancer related deaths. It is unclear, which factors contribute to this step. Recent studies indicate a crucial role of the tumor microenvironment in malignant transformation and metastasis. Studying cancer cell invasion and detachments quantitatively in the context of its physiological microenvironment is technically challenging. Especially, precise control of microenvironmental properties in vivo is currently not possible. Here, I studied the role of microenvironment geometry in the invasion and detachment of cancer cells from the bulk with a simplistic and reductionist approach. In this approach, I engineered microfluidic devices to mimic a pseudo 3D extracellular matrix environment, where I was able to quantitatively tune the geometrical configuration of the microenvironment and follow tumor cells with fluorescence live imaging. To aid quantitative analysis I developed a widely applicable software application to automatically analyze and visualize particle tracking data. Quantitative analysis of tumor cell invasion in isotropic and anisotropic microenvironments showed that heterogeneity in the microenvironment promotes faster invasion and more frequent detachment of cells. These observations correlated with overall higher speed of cells at the edge of the bulk of the cells. In heterogeneous microenvironments cells preferentially passed through larger pores, thus invading areas of least resistance and generating finger-like invasive structures. The detachments occurred mostly at the tips of these structures. To investigate the potential mechanism, we established a two dimensional model to simulate active Brownian particles representing the cell nuclei dynamics. These simulations backed our in vitro observations without the need of precise fitting the simulation parameters. Our model suggests the importance of the pore heterogeneity in the direction perpendicular to the orientation of bias field (lateral heterogeneity), which causes the interface roughening

Type I interferon immune signaling in murine systematic candidiasis

Pilzartige Sepsis ist die häufigste Todesursache bei Krankenhausaufnahmen und Candida ist dabei das häufigst isolierte Pathogen. Systemische Candida Infektionen zeigen eine Mortalitätsrate von etwa 30-40% bei immunkomprimierten Patienten. Candida glabrata ist ein hefeartiger pathogener Pilz, der sich als die zweit-häufigste Ursache der systemischen Candidiasis entwickelt hat. Typ I Interferone (Typ I IFN) sind wichtige Regulatoren des angeborenen Immunsystems. Obwohl unser Verständnis über die Rolle von den Typ I IFN bei viralen und bakteriellen Erkrankungen sich verbessert hat, ihre Rolle bei pilzerkrankungen wird erst neu ausführlich beschrieben. Unsere Gruppe hat schon publiziert, dass die Candida Spezies bei konventionellen dendritischen Zellen durch die Erkennung der fungalen Nukleinsäuren von TLR7 (Mustererkennungsrezeptor) die Typ I IFN induzieren. Außerdem tragen die Typ I IFN bei der Ausdauer der systemischen Infektion mit C. glabrata in Mäusen bei. Hier haben wir die Rolle von Typ I IFN in murinen systemischen C. glabrata Infektionen in Milz weiter untersucht. Wir haben gezeigt, dass die Rolle von Typ I IFN bei der Entfernung des Pathogens organ- und zeitabhängig ist, wobie die Typ I IFN die Ausdauer der Infektion bei spätern Stadien fördern. Am 7. Tag der Infektion waren die Milze vergrößert von IFNAR1-/- Mäusen und zusätzlich war die transkriptionelle Antwort stärker. Durch die Anwendung von Clustering-Methoden und mit dem NodeFinder (ein neues Computer-Programm, womit wir die Gene in koregulierten Netzwerken gruppiert haben) haben wir mehrere wichtige beteiligte Mechanismen entdeckt. Immunität-bezogene GTPasen waren höher exprimiert in IFNAR1-/--Milz am 3. Tag. Merkwürdigerweise stehen diese GTPasen unter Typ II IFN (IFN-γ) Regulation. Zusätzlich waren andere von Typ II IFN-regulierten Gene zum selben Zeitpunkt der Infektion (und 1. Tag) in der IFNAR1-/--Milz höher exprimiert. Interessanterweise waren antimikrobielle und inflammatorische Gene höher exprimiert am 7. Tag. Zudem waren mehr inflammatorische Monozyten während der Infektion und auch mehr Makrophagen im Stillstand in WT-Milz rekrutiert. Die Phagozytose und tötende Eigenschaften von Makrophagen waren nicht betroffen in vitro. Unsere Befunde stellen die wichtige Rolle der Typ I IFN bei der systemischen Candidiasis in Mäusen dar.Fungal sepsis is the most frequent cause of death between hospitalized human patients and Candida spp. being the most frequent cause among. Disseminated candidiasis is a life-threatening disease in immunocompromised individuals with a mortality rate of around 30-40%. The yeast-like fungus Candida glabrata has emerged as the second most frequent cause of systemic candidiasis. Type I interferons (IFNs) are important modulators of the innate host immunity. Although our understanding about role of type I IFNs in bacterial and viral infections has improved, their role in fungal infections are recently being revealed. Our group has previously reported that Candida spp. induce type I interferon (IFN) response in conventional dendritic cells via recognition of Candida nucleic acids the TLR7 pattern recognition receptor. Moreover, type I IFNs also contribute to fungal persistence in a murine model of systemic candidiasis. Here, we have further investigated the role of type I IFNs in murine systemic C. glabrata infections. We show that type I IFNs influence pathogen clearance in an organ- and timely-dependent manner, where type I IFN signaling contributes to persistence at later stages of infection. Spleens were more enlarged in the absence of type I IFN signaling at day 7. Additionally, the transcriptional response was also higher at day of IFNAR1-/- spleens. Also our data suggest a delay or absence in resolution of immune responses. Using clustering techniques and with NodeFinder, novel software tool we have created to group genes into co-regulated network, identified several important pathways involved. Immunity-related GTPases were higher expressed in IFNAR1-/- at day 3 of the infection. Noteworthy, these GTPases were also IFN-γ inducible. Interestingly, another group of genes under type II IFN (IFN-γ) regulation was also higher expressed in IFNAR1-/- spleens. Importantly, genes with antimicrobial inflammatory properties were also higher expressed in IFNAR1-/-. Moreover wildtype (WT) spleens were recruiting more inflammatory monocytes upon infection and more macrophages in steady state. Yet macrophage phagocytosis and killing were not affected by type I IFNs in vitro. Taken together, our work demonstrates on important role of type I IFN signaling in a murine model of invasive Candidemia

The RNA-binding protein tristetraprolin schedules apoptosis of pathogen-engaged neutrophils during bacterial infection

Protective responses against pathogens require a rapid mobilization of resting neutrophils and the timely removal of activated ones. Neutrophils are exceptionally short-lived leukocytes, yet it remains unclear whether the lifespan of pathogen-engaged neutrophils is regulated differently from that in the circulating steady-state pool. Here, we have found that under homeostatic conditions, the mRNA-destabilizing protein tristetraprolin (TTP) regulates apoptosis and the numbers of activated infiltrating murine neutrophils but not neutrophil cellularity. Activated TTP-deficient neutrophils exhibited decreased apoptosis and enhanced accumulation at the infection site. In the context of myeloid-specific deletion of Ttp, the potentiation of neutrophil deployment protected mice against lethal soft tissue infection with Streptococcus pyogenes and prevented bacterial dissemination. Neutrophil transcriptome analysis revealed that decreased apoptosis of TTP-deficient neutrophils was specifically associated with elevated expression of myeloid cell leukemia 1 (Mcl1) but not other antiapoptotic B cell leukemia/ lymphoma 2 (Bcl2) family members. Higher Mcl1 expression resulted from stabilization of Mcl1 mRNA in the absence of TTP. The low apoptosis rate of infiltrating TTP-deficient neutrophils was comparable to that of transgenic Mcl1-overexpressing neutrophils. Our study demonstrates that posttranscriptional gene regulation by TTP schedules the termination of the antimicrobial engagement of neutrophils. The balancing role of TTP comes at the cost of an increased risk of bacterial infections

WASp triggers mechanosensitive actin patches to facilitate immune cell migration in dense tissues

When crawling through the body, leukocytes often traverse tissues that are densely packed with extracellular matrix and other cells, and this raises the question: How do leukocytes overcome compressive mechanical loads? Here, we show that the actin cortex of leukocytes is mechanoresponsive and that this responsiveness requires neither force sensing via the nucleus nor adhesive interactions with a substrate. Upon global compression of the cell body as well as local indentation of the plasma membrane, Wiskott-Aldrich syndrome protein (WASp) assembles into dot-like structures, providing activation platforms for Arp2/3 nucleated actin patches. These patches locally push against the external load, which can be obstructing collagen fibers or other cells, and thereby create space to facilitate forward locomotion. We show in vitro and in vivo that this WASp function is rate limiting for ameboid leukocyte migration in dense but not in loose environments and is required for trafficking through diverse tissues such as skin and lymph nodes

Cellular locomotion using environmental topography

Eukaryotic cells migrate by coupling the intracellular force of the actin cytoskeleton to the environment. While force coupling is usually mediated by transmembrane adhesion receptors, especially those of the integrin family, amoeboid cells such as leukocytes can migrate extremely fast despite very low adhesive forces1. Here we show that leukocytes cannot only migrate under low adhesion but can also transmit forces in the complete absence of transmembrane force coupling. When confined within three-dimensional environments, they use the topographical features of the substrate to propel themselves. Here the retrograde flow of the actin cytoskeleton follows the texture of the substrate, creating retrograde shear forces that are sufficient to drive the cell body forwards. Notably, adhesion-dependent and adhesion-independent migration are not mutually exclusive, but rather are variants of the same principle of coupling retrograde actin flow to the environment and thus can potentially operate interchangeably and simultaneously. As adhesion-free migration is independent of the chemical composition of the environment, it renders cells completely autonomous in their locomotive behaviour