The Huang laboratory has recently established a powerful experimental system by genetically capturing chandelier cells (ChCs), interneurons that exert decisive control over pyramidal neuron (PyN) firing. We aim to establish a cell type-based experimental paradigm that will longitudinally integrate key developmental steps in the larger context of generation and integration of functional ChCs into telencephalic circuits. Genetic analysis of ChCs will provide a key entry point to understanding circuit assembly and help understand the etiology of neuropsychiatric disorders. ChCs regulate neural ensembles in major telencephalic structures such as neocortex, hippocampus, piriform cortex and amygdala. We will study the ventral telencephalic source of ChCs to identify possible spatially or temporally distinct progenitors which generate ChCs for different regions. We will extend our analysis to identify distinct progenitor pools and the mechanisms which generate different neocortical ChC subtypes based on specific laminar location (layers 2, 5 and 6). Major events in cortical ChC development have been described and we will examine the mechanisms of migration of ChCs at a cellular level, particularly the role of cortical PyNs in recruiting ChCs to specific layers. Using genetic engineering, fate mapping, imaging and electrophysiology, we will achieve a high resolution description of key events, and begin to explore the underlying molecular mechanisms.

HIV relies heavily on remodeling host regulatory networks for its replication. One major target of this remodeling process is the cellular ubiquitin machinery, which modifies proteins by ubiquitin moieties to target them for degradation and to modulate their activities. HIV exploits the ubiquitination system as a means to interfere with crucial immune regulatory mechanisms in antiviral pathways and to facilitate its own propagation. Therefore, targeting the interactions between HIV proteins and the human ubiquitin machinery might be a promising direction for therapeutic intervention of HIV infection. Several viral proteins have been shown to interact with ubiquitin ligases to regulate the activity of both viral and host proteins. However, this interface remains, to a large extent uncharacterized in terms of the exact molecular composition of HIV-human ubiquitin ligase complexes, their substrates and the extended protein interaction network.
Here, I propose a strategy combining quantitative mass spectrometry (MS) and computational network analysis approaches to characterize protein-protein interactions (PPIs) and ubiquitination events between HIV-1 proteins and the host ubiquitin ligases in the context of HIV-1 infection. Ubiquitin ligase-substrate relationships revealed by this strategy will be validated using a ligase trapping approach and their influence on the virus infection will be characterized by RNAi. I expect that this project will reveal new ubiquitin ligase-substrate relationships relevant for HIV-1 infection, identify specific targets for therapeutic interventions, and provide new insights into the role of the human ubiquitin machinery in immune response.

Emerging evidence suggests that GABAergic inhibitory circuits play critical roles in Rett Syndrome (RTT) pathology. The balance of excitation and inhibition (E/I balance) is complexly altered in the MeCP2 mutant mice. While synaptic dysfunctions in RTT are highly circuit dependent, various in vitro and in vivo studies have found different effects on different cell types in MeCP2 KO mice. Therefore, the precise nature of circuit abnormality in RTT and the contribution of different cell types remain unclear. One intriguing possibility is that MeCP2 deletion affects almost all neuron types, but may affect some neuron types more than others due to their developmental and integrative properties. Thus, one hypothesis is that different inhibitory types are differently impacted by MeCP2. Among the interneuron populations, soma-targeting parvalbumin (PV)-expressing neurons and dendrite-targeting somatostatin (SOM)-expressing neurons are the two major subclasses. SOM neurons integrate large-scale inputs and are crucial for feedback top-down modulation. They inhibit both pyramidal neurons and also PV neurons and hence complexly mediate E/I balance. PV neurons mainly subserve gain control and feedforward inhibition. The primary objective of this proposal is to examine how and to what degree SOM and PV neurons are affected in MeCP2 mutant mice, and whether MeCP2 deletion in SOM or PV neurons in a cell-autonomous manner leads to RTT pathology. I will also determine whether selective activation of SOM or PV neurons by optogenetic approach rescues behavioral abnormalities in MeCP2 mutant mice.

The aim of this project is to obtain the first high-resolution structure of a human receptor of the G-protein coupled receptor (GPCR) adhesion family, GPR56. This receptor has a unique N-terminal domain which is autocatalytically cleaved and subsequently reattached to the receptor, mediating interactions between the extracellular matrix and intracellular pathways within the cell. A number of studies have implicated a role of GPR56 in cancer where adhesion and detachment play important roles in tumor formation and migration. GPR56 is overexpressed in a number of human cancer forms including glioma and pancreatic cancer. In addition, mutations in GPR56 gives rise to a rare inherited disease causing cortical malformation in humans, further establishing the importance of GPR56 in the human body.
The applicant will conduct research in the laboratory of Prof. Stevens which specializes in determination of human GPCR structures. The applicant will use several techniques including codon optimization, insect cell expression and crystallization in lipidic phases. x-ray free electron laser and conventional synchrotron radiation will be used to obtain a high-resolution structure of GPR56 which could pinpoint a putative ligand binding site. Furthermore, the possibility of oligomerization of the receptor in the membrane will be investigated using fluorescent methods which, in combination with structural data, could give insight into the broad ligand-binding capabilities of this receptor.

Primary cilia are microtubule-based organelles that project from the cell surface. Primary cilia are well established as important sensory and signaling structures, in which a plethora of signaling receptors, such as G protein-coupled receptors (GPCRs), are concentrated. A dysfunction of primary cilium results in several manifestations called a “ciliopathy”. The deficiencies include retinal degeneration, polycystic kidneys, and obesity, where the pathogenesis mechanisms remain largely unknown. While several classical studies indicated the relationship between ciliopathy-related obesity and leptin resistance in neurons, a very recent study clearly showed that the leptin-resistance arises as a consequence of obesity. Another unexplored mechanism that could contribute to obesity would be a defect in adipogenesis. In this study, I will try to verify this hypothesis. In one branch of the study, I will identify the ciliary GPCR that controls adipogenesis in 3T3-L1 preadipocytes, based on its ability to differentiate preadipocytes, ciliary localization, and dependence on the BBS and Tubby pathways, both of which regulate GPCR-trafficking and body weight control. To verify the relationship between the defect in the GPCR signaling and increased fat pad in vivo, I will examine the transplantation of preadipocytes with or without the GPCR in athymic mice. Moreover, I will identify the downstream signaling of the GPCR by gene expression-based, localization, and protein network analysis.
Collectively, this study will provide the new knowledge of the signaling pathway in primary cilia, and critical insight into how the deregulation of this signaling causes obesity.

The main aim of the proposed research project is to elucidate the key principles underlying sensory processing in visual cortex. The central hypothesis is that sensory perception is an active process based on predictions and the detection of deviations from these predictions. In other words, much of what we perceive is not the result of what our sensory organs transmit to our brains but either the result of what we expect to perceive or the result of a large deviation from these expectations. Such predictions of sensory feedback can either be based on motor-related signals (e.g. in the form of an efference copy of motor commands), or on memory-related signals. We could recently show that mouse primary visual cortex receives input from both the anterior cingulate cortex and the hippocampal formation. These two inputs could serve as substrates for motor and memory based predictions. I now propose to characterize these two pathways both anatomically and functionally. We will use viral and genetic techniques to reconstruct the detailed anatomy of both connections, and use axonal and cellular imaging of calcium dynamics during behavior to characterize motor and memory related input signals to visual cortex.

Genetic information can be epigenetically modified independently of the DNA sequence itself. These chromatin-based regulatory mechanisms play a fundamental role in many biological processes, for example differentiation and development. Despite recent advances in mapping epigenetic states genome-wide, a mechanistic understanding of how they affect gene expression and the transcription machinery is missing. During Dosage compensation (DC), the expression of the X chromosome is equalized between females (XX) and males (XY). As DC operates independently of the DNA sequence, it provides an ideal system to study transcriptional regulation by epigenetic mechanisms. In Drosophila, DC operates on the male X by histone acetylation and recruitment of the MSL complex. I plan to address, how the MSL complex affects transcription by studying the biochemical and biophysical properties of a fully recombinant complex in the context of designer chromatin. For this, I will take advantage of recently developed techniques to produce homogenously modified histones. I then plan to establish a highly purified in vitro transcription system to analyze, whether and how histone modifications and the MSL complex affect transcription. Third, I would like to study whether co-transcriptional processes such as RNA processing are enhanced for X-linked genes as part of the DC process in vivo. Here, I foresee to discover potentially novel crosstalks between the DC machinery, transcription and chromatin. Altogether, these studies will elucidate the mechanistic basis of modulated expression during DC and also provide important progress in understanding how the epigenome affects the transcription machinery.

Decisions can arise from multiple systems in the brain, which can be dissociated behaviorally, neurally, and computationally. Much progress has been made in our understanding of the “Pavlovian” and “habit” systems, but the neural mechanisms underlying the “planning” system remain elusive. An emerging model system for the study of planning at the neurobiological level is the recording and manipulation of “place cells” in the hippocampus of rats solving navigation problems. Sequences of place cell activity can be decoded to reveal signatures of a planning process: possible trajectories are represented serially at decision points and following reward receipt, and include constructed, never-experienced trajectories. However, computational work clearly shows that serial, brute-force search will fail in complex environments. Accordingly, many efficient algorithms for approximate search have been developed in artificial intelligence (AI) and reinforcement learning (RL), but the algorithms used by the brain to plan in complex environments are unknown.
We propose to elucidate the neural mechanisms of planning in complex environments through the recording, manipulation, and simulation of hippocampal activity, afforded by interdependent approaches rooted in neurobiology, engineering, and computational modeling. We will record place cell activity on-line during planning and off-line during consolidation periods, on tasks sufficiently complex to require approximate planning methods, and that satisfy established behavioral criteria for planning. AI and RL-derived simulations will be applied to these tasks to generate contrasting predictions about the content and frequency of hippocampal sequences, and provide a computational basis for targeted manipulations. To test the impact of hippocampal sequences on associated circuits and behavior, we will develop real-time methods for sequence decoding, enabling the disruption and reinforcement of specific sequences with electrical and optogenetic tools. The combination of these approaches permits the disentangling of interactions between fast, “on-line” planning and the slower “structure learning” of appropriate task representations. The proposed work will reveal the real-time dynamics and associated computational principles that underlie navigational planning in complex environment, and uniquely links the neural circuit, representational and computational levels of a model planning system.

The overall goal of this proposal is to study the relationship between characteristics of functional brain networks and behavior. We will use a combination of imaging and graph theoretical approaches to create and analyze functional brain networks generated during memory formation and retrieval followed by targeted optogenetics to manipulate identified network hubs. At the behavioral level we will use fear conditioning to generate memories. To permanently tag neurons activated during memory acquisition we will use the recently developed TRAP (targeted recombination in active populations) mouse line in which expression of Cre recombinase is controlled by the neuronal activity regulated c-fos promoter. We will tag neurons active during fear memory acquisition with a genetically encoded fluorescent marker. After memory retrieval brains, will be processed for imaging using the CLARITY technique. Functional networks will be built by examining the co-variation of the number of neurons expressing the fluorescent marker (acquisition) or elevated levels of activity regulated genes such as c-fos (retrieval) throughout the brain. Graph theoretical analysis will be used to identify brain regions with high interconnectivity (i.e, hubs). We will interrogate the network using targeted optogenetics in which adeno associated viruses expressing inhibitory opsins in a Cre recombination dependent manner are infused into hub brain regions. We will examine the effect of inhibiting hubs during testing on memory retrieval and the resulting functional network. In this way we will be able to draw causal inferences between characteristics of functional brain networks and memory expression.

During the past 40 years, behavioral studies have confirmed the presence of magnetoreception in all major groups of Vertebrates, including fish, amphibians, reptiles, birds, and mammals, as well as in insects, crustaceans, and in a variety of microorganisms including the magnetotactic bacteria and protists. Electrophysiological studies in birds and fish indicate that the ophthalmic branch of the trigeminal nerve is one of the conduits of magnetic field information to the brain, implying that this pathway evolved before the last common ancestor of Vertebrates; more recent work suggests the vestibular system in birds might also be involved. Other work suggests magnetoreception might also arise in the retina, perhaps through optical effects on a protein like cryptochrome.
Despite this widespread presence in animals, the question of whether or not humans possess a remnant of these ancient magnetosensory systems has remained an extraordinarily controversial issue. Humans possess both biological magnetite, as well as cryptochromes, arguing that magnetoreception is a physical possibility. However, reports of magnetic fields influencing human orientation, made by a British group 30 years ago, failed the ‘acid test’ of Science when the initial orientation effects could not be repeated, thereby precluding tests of the magnetic influence. In contrast, more recent and as yet preliminary results from psychological experiments by the Kirschvink group support the hypothesis that some human subjects may have a subconscious remnant of this magnetic sensory system. Using our combined geophysical, psychological, and engineering skills, our goals for this HFSP project are to attempt verification, improve the power of the procedures, map the neural circuits underlying the sensitivity, and ultimately develop behavioral-neural techniques to bring human magnetoreception into conscious awareness. If successful, these tools could also be applied to test biophysical hypotheses for the underlying transduction mechanism(s). As a significant byproduct, the psychophysical and engineering techniques developed will lead to more interactive BMI (Brain Machine Interface). It will be applicable to any other cases where a human brain has potentially plastic sensory abilities but not revealed yet.

Cell size is one of the most fundamental aspects that define the properties of a cell. During cell division, daughter cell size is determined by the position of the mitotic spindle, which is controlled by dynein-based cortical forces that pull on the spindle. During my HFSP fellowship, I identified cortical dynein regulatory systems that ensure equal-sized cell division in HeLa cells. However, the biological significance of equal-sized cell division has not been studied. For example, maintenance of cell size may play an important role in stem cell self-renewal.

To precisely control spindle position in a variety of cell types, I will develop a light-inducible system in which cortical dynein regulators are targeted to locally illuminated regions to manipulate spindle position in a spatially and temporally controlled manner. This optogenetic approach represents a powerful tool that will allow me both to understand the molecular functions of targeted proteins, and to manipulate spindle position to generate unequal-sized divisions in cell types that normally divide symmetrically.

Using the optogenetic system in parallel with RNAi, I will analyze the significance of symmetric cell division in differentiated cells and embryonic stem cells.

In this proposal, I will:

1. Develop an optogenetic-based approach to manipulate spindle position

2. Define the roles of symmetric cell division in HeLa and non-transformed Rpe1 cells

3. Analyze the mechanisms and roles of symmetric cell division in self-renewing ES cells.

The support of the CDA is critical for allowing me to establish my independent research and will enable me to assemble a team to conduct these experiments.

In all domains of life ribosomes catalyze protein synthesis. To ensure that these large molecular machines are correctly assembled, a plethora of protein factors are deployed in eukaryotes, which facilitate the assembly pathway in which 4 ribosomal RNAs and 79 proteins are integrated into a fully functional ribosome. Defects in ribosome assembly are associated with several serious human diseases, which underline the central importance of this process.

The initial transcription of pre-ribosomal RNA (35S pre-rRNA in yeast, containing 18S, 5.8S and 25S rRNA) is followed by a hierarchical assembly of a giant complex termed the Small Subunit Processome (the 2.2 Megadalton SSU Processome) from several multi-protein sub-complexes. This complex is unique to eukaryotes and most of its constituents are essential proteins. While many of the protein components of the SSU Processome have been identified, our understanding of this essential complex is currently limited by a lack of structural and mechanistic insights.

This proposal aims at the structural and mechanistic characterization of the SSU Processome. To unravel its functional architecture an integrated structure-function approach will be applied, which combines yeast genetics, novel biochemical tools as well as complementary structural studies using X-ray crystallography and cryo-electron microscopy. These approaches will be used synergistically to elucidate the inner workings of the SSU Processome during eukaryotic ribosome assembly.

Proliferating cells must grow and duplicate their contents before dividing. The cell cycle comprises the set of regulatory interactions that protect against improper division by imposing a strict order on the processes leading up to cytokinesis. A key aspect of this control is the coordination of overall growth with division to maintain a consistent cell size across populations and generations. In S. cerevisiae this is achieved by delaying progression through the cell cycle in G1 phase until the cell has reached a minimum size. Although genetic screens and in-depth studies have identified a set of proteins affecting cell size regulation, the molecular mechanism that allows cells to measure their size is still obscure. One major candidate for a size sensor is the unstable G1 cyclin Cln3, which triggers irreversible commitment to cell division. Since Cln3 concentration is thought to remain constant through G1, we hypothesize that cells may titrate the number of Cln3 molecules, which increases proportionally with cell size, against their DNA, which remains constant in G1. Such a mechanism could result in an enzyme activity proportional to the number of Cln3 molecules rather than to their concentration. Here, we propose to test this hypothesis both in vitro and in vivo. First, we will determine if the phosphorylation of key Cln3-Cdk1 substrates is affected by the presence of DNA in vitro. Second, using single cell quantitative microscopy,we will confirm the relevance of DNA-dependent phosphorylation to size control in vivo. Demonstrating that DNA dependence of Cln3-Cdk1 substrate phosphorylation is required for size control would directly support the titration model of cell size control and thereby solve a longstanding question in biology.

Synthetic biology offers the possibility of new therapies based on functional cellular implants to cure intractable diseases, such as immune diseases and metabolic diseases. For example, the need for diabetics to inject insulin after every meal could potentially be avoided by introducing functional cellular implants that rapidly release insulin on demand. This would dramatically improve patients’ quality of life. However, it is difficult to rapidly control protein secretion in response to stimulation because current artificial cellular functions rely mainly on slow transcriptional control of transgenes within the cells.
My proposed project aims to create new cellular functions capable of rapidly modulating biological systems by secreting effector molecules outside the cells in response to external stimuli. It is established that a well-regulated exocytosis pathway exists in a wide variety of cells and can be artificially reconstructed in artificial liposomes. Therefore the project will focus on creating new, rapidly responsive cellular functions by artificially modulating the exocytosis pathway in living cells.
I intend to validate this design approach for innovative future cell-based therapies by constructing a new therapeutic model. Specifically, I propose to construct a model system for treating rejection after transplantation without the need for administration of immunosuppressive drugs, by incorporating artificial cellular functions into the surface layer of the transplant. These functions will serve to sense contact of active T cells and to rapidly secrete T-cell-blocking effector molecules only in the immediate vicinity of the transplant.

Misfolding diseases arise as a consequence of accumulation of undesired proteins in the cell, which is due to failure of protein quality control system. The ubiquitin proteasome system, involved in this control, degrades a large number of cellular proteins and dysfunction of the proteasome has been associated with a number of misfolding diseases. Thus, rescuing the defects resulting from proteasome inhibition could be of a therapeutic use. The aim of this project is to identify strategies to rescue from proteasome impairment. Recent unpublished work in the host laboratory has led to the identification of a potent and previously unidentified suppressor of proteasome dysfunction in yeast. Since the function of the proteasome is evolutionary conserved it is likely that a protein of similar function exists in mammals. The first goal of my project is to identify this homologue. In addition, the host lab has recently discovered that cells can survive with undegraded proteins that accumulate as a result of proteasome inhibition when supplemented with amino acids. I will determine whether amino acid supplementation can rescue deleterious consequences of protein misfolding in an in vivo model of a misfolding disease. In summary, this research can lead to better understanding of proteasome assembly and function as well as highly impact misfolding diseases field.

Combine the well established RNA switch in the laboratory of Dr. Hirohide Saito at Kyoto University with cytotoxic supramolecular nanofibers from the laboratory of Dr. Bing Xu at Brandeis University, the objective of the proposed research is to design a two-component device for selectively elimination of cancer cells based on the recognition of over-expressed RNA binding proteins (RBPs) in cancer cells and the degradation controlled cytotoxicity of molecular nanofibers. To achieve the objective, the proposed research will design RNA switches that response to RBPs, such as Sam68, eIF4E, La, or HuR (L. Wurth, Comp. Funct.Genom., 2012), that are over-expressed in cancer cells. The recognition of RBPs in cancer cells prohibits the expression of downstream mRNA that codes for esterase. On the contrary, low expression of RBPs in normal cells allows the expression of esterase. Therefore, the toxic nanofibers, which can be degraded into nontoxic fragments by the esterase, will only induce cytotoxicity in cancer cells, while leaving the normal cells unharmed. The proposed research innovatively combines synthetic biology with supramolecular chemistry, by which both precise recognition and elimination of cancer cells can be achieved. Importantly, the two-component device unlikely would induce adverse side effect on normal cells and tissues, as the device, by itself, is nontoxic and toxicity appears only under the recognition of cancer marker RBPs. The high tolerance of the device by normal cells and tissue not only may lead to a new approach for cancer therapy, but also will contribute to the research in iPS cells for the removal of cancer cells from iPS cell cultures.

The origin and maintenance of new species is one of the key questions in evolutionary biology. Yet, little is known about the genetic and epigenetic basis of speciation, especially outside of model organisms. In conventional models of speciation, the genetic architecture of diverging species will become so different that genes or genomic regions are incompatible if brought together in hybrids. A recent idea is that a broad variety of epigenetic mechanisms, like methylation, may also play a role in the hybrid phenotype. This project will investigate the role of methylation and gene expression in hybrid incompatibilities. For this, I will use the recently speciated Formica rufa group ants and their hybrids. First, I will study gene expression and methylation patterns genome-wide by RNA-seq and whole genome bisulfite sequencing and test if gene expression and methylation are disrupted in hybrids. Second, I will test the hypothesis that genomic regions implicated in hybrid incompatibilities are associated with divergent or disrupted methylation and/or expression patterns. The novelty of this project lies in combining genome-wide data on genetic incompatibilities, methylation and expression. This project will generate new data on the epigenetic basis of speciation, a promising research area where only a few studies exist at present. Furthermore, the project will widen my scientific and analysis skills by learning me to master genome-wide sequence data. The results will shed important insight into genetic and epigenetic basis of speciation, how methylation coordinates gene expression and the mechanisms that underlie hybrid dysfunction and novelty.

Cell surface receptors, transporters, and ion channels underlie communication between the intracellular and extracellular environments. Cell surface receptors exist in several stable states and likely sample multiple conformations during transitions between these states. Yet, fundamental mechanisms relating these conformational changes to protein function remain poorly understood. Single molecule studies have revealed dynamics of purified proteins, transplanted out of their cellular environment, but existing single molecule methods lack the sensitivity to examine protein dynamics in live cells. Moreover, many important membrane proteins cannot be purified and reconstituted, making them impossible to study with existing single molecule methods. The proposed research will extend single-molecule measurements to these previously inaccessible proteins. Our studies will utilize plasmonic nanostructures that will enable spatial isolation of proteins within nanoscale ‘chambers’, by growing cells on arrays of these tiny apertures. While the optical confinement of single receptors in these apertures will virtually eliminate background fluorescence, the plasmonic antenna will enhance the fluorescence emission of dyes and increase photostability. These cooperative features will significantly improve detection efficiency and allow for the resolution of single protein dynamics in live cells.
We will couple this innovative live cell, single molecule, imaging system with patch clamp electrophysiology that will enable precise control of voltage-gated ion channel gating. This will allow us to examine conformational dynamics of receptors at sub-millisecond time scales using single-molecule FRET. We will apply this new technique to measure conformational changes in Kv1.2 voltage gated potassium channels which are essential electrical regulators in the nervous system. Applied changes in membrane voltage trigger structural rearrangements within these ion channels, culminating in opening of the channel pore. The proposed studies will allow us to detect these conformational changes in single ion channels, and directly correlate conformational changes to receptor function. This collaborative and multidisciplinary project has the potential to establish a novel platform for a broad range of single-molecule applications in live cells.

Although conserved ageing pathways have been studied in invertebrate model systems, the links between them are still poorly described. Medical interventions for human ageing are likely to use adult stem cells (ASCs) as therapeutic agents. Thus, it would seem timely to utilize model systems that have ASCs to understand how ASCs respond to one of the key drivers of ageing, oxidative stress. DNA replication causes the chromosomes to shorten as cells divide unless telomerase activity counteracts this process. Also, as animals increase in chronological age, oxidative damage would contribute to telomere shortening that slows down tissue’s homeostatic turnover. However, evidences have suggested other telomere-independent functions of telomerase that arise during oxidative stress to protect cells from oxidative damage. I aim to investigate how ASCs in ageing and non-ageing flatworms cope with oxidative stress and regulate reactive oxygen species (ROS) homeostasis. Telomere maintenance and telomerase activity are adapted to asexual reproduction, such that they allow the avoidance of ASCs senescence by telomere shortening. I will use flatworms with sexual and asexual biotypes to understand how they avoid ageing when challenged by ROS. I will study how telomerase activity is regulated, how this affects stem cell maintenance and whether telomerase exerts protective functions on mitochondria to prevent mitochondria dysfunction during oxidative stress. This study will yield unprecedented insights into biological immortality, regenerative capacity and ageing, making use of powerful flatworm models to understand the links between oxidative stress and telomerase in the context of ASCs.

Our present concept of CRL function derives mainly from scrutinized SCF and APC/C complexes that drive cell cycle progression by targeted proteolysis of their substrates. However, accumulating evidence highlights vital roles for CRL3 and CRL4 in orchestrating mitotic events. To further illuminate the function of these CRLs during cell division we need to (i) identify important adaptors and their critical substrates, (ii) decipher the biological output of individual ubiquitination reactions as well as (iii) extend our knowledge of general CRL enzymology and regulation. As of yet the identification of substrates for specific E3 ligases has been technically challenging. This primarily reflects short substrate residence times, rapid turnover of ubiquitinated substrates destined for degradation and often low cellular abundance. Here, I will pursue a novel strategy of a single molecule TIRF microscopy-based flow-cell assay tailored to overcome these limitations. In line with our research priorities this assay supports fast compositional probing of novel CRL complexes and ultrasensitive detection of prevalently transient and rare physiological E3 ligase–substrate encounters from cell extracts. On top of that, I will exploit this single-molecule imaging technique to recapitulate and quantitatively analyze mechanistic aspects of dynamic E3 ligase function. A list of novel putative mitotic Cul4 substrate specific adaptors that emerged from in silico predictions and high-content RNAi screening previously conducted in our lab provides the biological foundation for this study that includes a comprehensive analysis of novel CRL4 complexes in mammalian cells.