cell biology

Cytoskeletal connectors: Deciphering the fundamental mechanisms of cytoskeletal dynamics and transport

PROJECT SUMMARY The cytoskeleton is a dynamic network of filamentous structures, including microtubules and actin, that regulate essential cellular processes such as cell shape, growth, and signaling. Cytoskeleton also serves as tracks for molecular motors, which transport a variety of cellular cargoes, including organelles, macromolecules, and vesicles. These cargoes are linked to motors by specialized connector proteins. Disruptions in connector proteins are implicated in a range of neurodevelopmental and neurodegenerative diseases, as well as cancers. Despite their importance, these proteins continue to be understudied, primarily due to their perceived role as passive linkers and the technical challenges in working with them. However, recent discoveries suggest that connector proteins may play more active roles, in some cases even have enzymatic functions. This proposal aims to uncover mechanisms of connector protein functions through a detailed investigation of actin-microtubule and motor-cargo interactions. Actin and microtubules are linked by the spectraplakin family of large and evolutionarily conserved proteins, critical for neuronal development and differentiation. Recent discoveries of ATPase domains within these proteins suggest they may haves beyond simply linking cytoskeletal components. One goal of this proposal is to investigate the role of spectraplakin’s ATPase domains via structural, biochemical, and cell biology approaches. Another goal is to explore how dynamic changes in motor-cargo connectors facilitate the transport of diverse cargoes along microtubule tracks. The focus will be on the cytoplasmic dynein-1 (dynein) and the connectors (adaptors) that activate and link dynein to cargo. Dynein is a microtubule minus-end directed motor that plays essential roles in cell division, and transports hundreds of different cellular cargoes. While several motor-cargo connectors have been identified, the regulatory mechanisms enabling cargo transport are not fully understood. We are investigating whether connector proteins work together to activate dynein movement and/or facilitate cargo handoff between different dynein complexes. Using innovative approaches, including time- resolved cryo-EM, complex in-vitro reconstitutions, and live-cell imaging in induced neurons, we are uncovering critical mechanisms that govern cytoskeletal connector proteins, furthering our understanding of how the cytoskeleton regulates essential cellular processes.

Developing a novel technology for studying T cell differentiation in vivo

Summary CRISPR-based genetic screens have revolutionized our understanding of gene functions and molecular mechanisms across various biological processes. In the field of T cell biology, CRISPR screens have played a pivotal role in identifying genes that impact critical aspects, such as T cell development, differentiation, and function. However, traditional screens have struggled to distinguish genes with diverse mechanisms of action, necessitating further investigations. To address this challenge, researchers have harnessed the power of CRISPR screens combined with single-cell sequencing (scCRISPR-seq), enabling the simultaneous assessment of genetic perturbations and high-dimensional phenotypes at the single-cell level. While scCRISPR- seq has predominantly been performed in vitro using immortalized cell lines, its physiological relevance is limited due to oversimplified biological context and disparities compared to primary cells. This limitation highlights the urgent need for large-scale in vivo scCRISPR-seq with primary T cells. However, various challenges have discouraged its widespread adoption. The use of viral vectors for sgRNA delivery compromises physiological relevance, as the in vitro activation conditions fail to faithfully represent the intricate T cell priming process in vivo. Moreover, viral vector components and continuous Cas9 expression can trigger immunogenicity and cytotoxicity, leading to cell depletion and hindering long-term studies. Additionally, current scCRISPR-seq methods face technical limitations, including low editing efficiency and inadequate perturbation identity recovery rates, which impede efficient large-scale in vivo applications. Fortunately, recent advances in ribonucleoprotein complex (RNP) transfection have addressed many of these challenges. This cutting-edge technology enables efficient gene editing in primary T cells without the need for in vitro activation or permanent Cas9 expression. Leveraging the high editing efficiency of RNP transfection, the investigator’s team aims to develop a novel strategy for in vivo T cell CRISPR screens. This innovative approach involves arrayed RNP transfection and co- transfer of T cells that recognize the relevant antigens. Instead of traditional genetic barcodes, the strategy utilizes congenic markers (CD45.1/45.2 and CD90.1/CD90.2) from donor TCR transgenic T cells as "external barcodes." These markers facilitate the recovery of gene perturbation identity at the single-cell level through the application of CITE-seq. Importantly, this RNP-based strategy seamlessly integrates with existing single-cell sequencing protocols, enabling the comprehensive assessment of transcripts, epitopes, and chromatin accessibility simultaneously. To demonstrate the efficacy of this strategy, the team plans to develop two benchmarking approaches: RNP-CET-seq to investigate the role of TCR regulators in T cell exhaustion and RNP-CATE-seq to map the gene regulatory atlas of exhausted CD8 T cells. In summary, the proposed RNP- based scCRISPR-seq strategy overcomes the limitations of current approaches, enabling large-scale, multi- module in vivo genetic screens within a physiologically relevant context across various disease models.

How the presynapse forms and functions”

Nervous system function relies on the polarized architecture of neurons, established by directional transport of pre- and postsynaptic cargoes. While delivery of postsynaptic components depends on the secretory pathway, the identity of the membrane compartment(s) that supply presynaptic active zone (AZ) and synaptic vesicle (SV) proteins is largely unknown. I will discuss our recent advances in our understanding of how key components of the presynaptic machinery for neurotransmitter release are transported and assembled focussing on our studies in genome-engineered human induced pluripotent stem cell-derived neurons. Specifically, I will focus on the composition and cell biological identity of the axonal transport vesicles that shuttle key components of neurotransmission to nascent synapses and on machinery for axonal transport and its control by signaling lipids. Our studies identify a crucial mechanism mediating the delivery of SV and active zone proteins to developing synapses and reveal connections to neurological disorders. In the second part of my talk, I will discuss how exocytosis and endocytosis are coupled to maintain presynaptic membrane homeostasis. I will present unpublished data regarding the role of membrane tension in the coupling of exocytosis and endocytosis at synapses. We have identified an endocytic BAR domain protein that is capable of sensing alterations in membrane tension caused by the exocytotic fusion of SVs to initiate compensatory endocytosis to restore plasma membrane area. Interference with this mechanism results in defects in the coupling of presynaptic exocytosis and SV recycling at human synapses.

The cell biology of Parkinson’s disease: a role for primary cilia and synaptic vesicle pleomorphism in dopaminergic neurons

Self-perception: mechanosensation and beyond

Brain-organ communications play a crucial role in maintaining the body's physiological and psychological homeostasis, and are controlled by complex neural and hormonal systems, including the internal mechanosensory organs. However, the progress has been slow due to technical hurdles: the sensory neurons are deeply buried inside the body and are not readily accessible for direct observation, the projection patterns from different organs or body parts are complex rather than converging into dedicate brain regions, the coding principle cannot be directly adapted from that learned from conventional sensory pathways. Our lab apply the pipeline of "biophysics of receptors-cell biology of neurons-functionality of neural circuits-animal behaviors" to explore the molecular and neural mechanisms of self-perception. In the lab, we mainly focus on the following three questions: 1, The molecular and cellular basis for proprioception and interoception. 2, The circuit mechanisms of sensory coding and integration of internal and external information. 3, The function of interoception in regulating behavior homeostasis.

Pro-regenerative functions of microglia in demyelinating diseases

Our goal is to understand why myelin repair fails in multiple sclerosis and to develop regenerative medicines for the nervous system. A central obstacle for progress in this area has been the complex biology underlying the response to CNS injury. Acute CNS damage is followed by a multicellular response that encompasses different cell types and spans different scales. Currently, we do not understand which factors determines lesion recovery. Failure of inflammation to resolve is a key underlying reason of poor regeneration, and one focus is therefore on the biology of microglia during de- and remyelination, and their cross talk to other cells, in particular oligodendrocytes and the progenitor cells. In addition, we are exploring the link between lipid metabolism and inflammation, and its role in the regulation of regeneration. I will report about our recent progress in our understanding of how microglia promote regeneration in the CNS.

The Standard Model of the Retina

The science of the retina has reached an interesting stage of completion. There exists now a consensus standard model of this neural system - at least in the minds of many researchers - that serves as a baseline against which to evaluate new claims. The standard model links phenomena from molecular biophysics, cell biology, neuroanatomy, synaptic physiology, circuit function, and visual psychophysics. It is further supported by a normative theory explaining what the purpose is of processing visual information this way. Most new reports of retinal phenomena fit squarely within the standard model, and major revisions seem increasingly unlikely. Given that our understanding of other brain circuits with comparable complexity is much more rudimentary, it is worth considering an example of what success looks like. In this talk I will summarize what I think are the ingredients that led to this mature understanding of the retina. Equally important, a number of practices and concepts that are currently en vogue in neuroscience were not needed or indeed counterproductive. I look forward to debating how these lessons might extend to other areas of brain research.

How do protein-RNA condensates form and contribute to disease?

In recent years, it has become clear that intrinsically disordered regions (IDRs) of RBPs, and the structure of RNAs, often contribute to the condensation of RNPs. To understand the transcriptomic features of such RNP condensates, we’ve used an improved individual nucleotide resolution CLIP protocol (iiCLIP), which produces highly sensitive and specific data, and thus enables quantitative comparisons of interactions across conditions (Lee et al., 2021). This showed how the IDR-dependent condensation properties of TDP-43 specify its RNA binding and regulatory repertoire (Hallegger et al., 2021). Moreover, we developed software for discovery and visualisation of RNA binding motifs that uncovered common binding patterns of RBPs on long multivalent RNA regions that are composed of dispersed motif clusters (Kuret et al, 2021). Finally, we used hybrid iCLIP (hiCLIP) to characterise the RNA structures mediating the assembly of Staufen RNPs across mammalian brain development, which demonstrated the roles of long-range RNA duplexes in the compaction of long 3’UTRs. I will present how the combined analysis of the characteristics of IDRs in RBPs, multivalent RNA regions and RNA structures is required to understand the formation and functions of RNP condensates, and how they change in diseases.

Charting the Proteome Landscape of Diverse Synapses In Vivo

From the cell biology of synaptic plasticity to SFARI

2nd In-Vitro 2D & 3D Neuronal Networks Summit

The event is open to everyone interested in Neuroscience, Cell Biology, Drug Discovery, Disease Modeling, and Bio/Neuroengineering! This meeting is a platform bringing scientists from all over the world together and fostering scientific exchange and collaboration.

2nd In-Vitro 2D & 3D Neuronal Networks Summit

The event is open to everyone interested in Neuroscience, Cell Biology, Drug Discovery, Disease Modeling, and Bio/Neuroengineering! This meeting is a platform bringing scientists from all over the world together and fostering scientific exchange and collaboration.

Investigating the functional single-cell biology of SynGAP1 pathways

In vitro bioelectronic models of the gut-brain axis

The human gut microbiome has emerged as a key player in the bidirectional communication of the gut-brain axis, affecting various aspects of homeostasis and pathophysiology. Until recently, the majority of studies that seek to explore the mechanisms underlying the microbiome-gut-brain axis cross-talk relied almost exclusively on animal models, and particularly gnotobiotic mice. Despite the great progress made with these models, various limitations, including ethical considerations and interspecies differences that limit the translatability of data to human systems, pushed researchers to seek for alternatives. Over the past decades, the field of in vitro modelling of tissues has experienced tremendous growth, thanks to advances in 3D cell biology, materials, science and bioengineering, pushing further the borders of our ability to more faithfully emulate the in vivo situation. Organ-on-chip technology and bioengineered tissues have emerged as highly promising alternatives to animal models for a wide range of applications. In this talk I’ll discuss our progress towards generating a complete platform of the human microbiota-gut-brain axis with integrated monitoring and sensing capabilities. Bringing together principles of materials science, tissue engineering, 3D cell biology and bioelectronics, we are building advanced models of the GI and the BBB /NVU, with real-time and label-free monitoring units adapted in the model architecture, towards a robust and more physiologically relevant human in vitro model, aiming to i) elucidate the role of microbiota in the gut-brain axis communication, ii) to study how diet and impaired microbiota profiles affect various (patho-)physiologies, and iii) to test personalised medicine approaches for disease modelling and drug testing.

New Strategies and Approaches to Tackle and Understand Neurological Disorder

Broadly, the Mauro Costa-Mattioli laboratory (The MCM Lab) encompasses two complementary lines of research. The first one, more traditional but very important, aims at unraveling the molecular mechanisms underlying memory formation (e.g., using state-of-the-art molecular and cell-specific genetic approaches). Learning and memory disorders can strike the brain during development (e.g., Autism Spectrum Disorders and Down Syndrome), as well as during adulthood (e.g., Alzheimer’s disease). We are interested in understanding the specific circuits and molecular pathways that are primarily targeted in these disorders and how they can be restored. To tackle these questions, we use a multidisciplinary, convergent and cross-species approach that combines mouse and fly genetics, molecular biology, electrophysiology, stem cell biology, optogenetics and behavioral techniques. The second line of research, more recent and relatively unexplored, is focused on understanding how gut microbes control CNS driven-behavior and brain function. Our recent discoveries, that microbes in the gut could modulate brain function and behavior in a very powerful way, have added a whole new dimension to the classic view of how complex behaviors are controlled. The unexpected findings have opened new avenues of study for us and are currently driving my lab to answer a host of new and very interesting questions: - What are the gut microbes (and metabolites) that regulate CNS-driven behaviors? Would it be possible to develop an unbiased screening method to identify specific microbes that regulate different behaviors? - If this is the case, can we identify how members of the gut microbiome (and their metabolites) mechanistically influence brain function? - What is the communication channel between the gut microbiota and the brain? Do different gut microbes use different ways to interact with the brain? - Could disruption of the gut microbial ecology cause neurodevelopmental dysfunction? If so, what is the impact of disruption in young and adult animals? - More importantly, could specific restoration of selected bacterial strains (new generation probiotics) represent a novel therapeutic approach for the targeted treatment of neurodevelopmental disorders? - Finally, can we develop microbiota-directed therapeutic foods to repair brain dysfunction in a variety of neurological disorders?

Role of Tunneling Nanotubes (TNTs) in the spreading of amyloid proteins in neurodegenerative diseases

Aging Brain Initiative Symposium: Cellular & Molecular Mechanisms of Neurodegeneration

The Aging Brain Initiative is an ambitious interdisciplinary effort by MIT focusing on understanding neurodegeneration and efforts to find hallmarks of aging, both in health and disease. The Initiative is broad, made up of scientists in several areas, including systems neuroscience, cell biology, engineering and computational biology, with core investigators from the Departments of Biology, Brain & Cognitive Sciences, Biological Engineering, and Computer Science & Artificial Intelligence Labs. "The theme of this symposium is Cellular & Molecular Mechanisms of Neurodegeneration.

Role of DIAPH3 in neural stem cell biology and tumor development