Biology subjects

Lasker, K.

Publications and source records attributed to Lasker, K..

11 recordsLinked to original sources

bioRxiv2026

nELAVL phosphorylation by CDKL5 regulates inter-condensates composition and communication to promote experience-dependent maturation of the visual cortex

Mutations in Cyclin-Dependent Kinase-Like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), an X-linked neurodevelopmental condition. Through a phosphoproteomic screen, we identified the neuron-specific nELAVL family of RNA-binding proteins as direct activity-dependent substrates of CDKL5. In support of this regulatory axis, single-nuclei transcriptomics of Cdkl5 knockout (KO) cortices revealed an enriched reduction in activity-dependent mRNAs. Mechanistically, we show that nELAVL proteins undergo phase separation to form biomolecular condensates, the size of which is gated by CDKL5 phosphorylation. Loss of CDKL5 leads to enlarged nELAVL condensates, which exhibit reduced binding affinity for the target mRNA Fos, resulting in its accelerated degradation. This disruption extends to inter-condensate communication: phosphodeficient nELAVL show diminished interaction with P-bodies, which themselves become enlarged in CDD mutant iNeurons. Functionally, the absence of nELAVL phosphorylation recapitulates the deficits in experience-dependent visual function observed in Cdkl5 KO mice. Our findings establish a critical molecular mechanism by which CDKL5-mediated phosphorylation governs mRNA metabolism by tuning the properties of nELAVL condensates and their communication with other biomolecular condensates, ultimately promoting experience-dependent maturation of the visual cortex.

neuroscience↗

bioRxiv2024

Biomolecular Condensates can Induce Local Membrane Potentials

Biomolecular condensates are a ubiquitous component of cells, known for their ability to selectively partition and compartmentalize biomolecules without the need for a lipid membrane. Nevertheless, condensates have been shown to interact with lipid membranes in diverse biological processes, such as autophagy and T-cell activation. Since many condensates are known to have a net surface charge density and associated electric potential(s), we hypothesized that they can induce a local membrane potential. Using an electrochromic dye, we demonstrate that poly-lysine/ATP condensates induce a localized membrane potential in Giant Unilamellar Vesicles. This effect diminishes with increasing salt concentration and higher ATP-to-poly-lysine ratios, underscoring the key role of condensate charge. Numerical modeling of the condensate-membrane interface using an electro-thermodynamic framework supports our experimental findings and highlights parameters expected to play a key role in the effect. These results have broad implications for biological processes regulated by membrane potential, particularly in contexts such as neuronal signaling, where condensate interactions with membranes may play a previously unrecognized regulatory role.

biophysics↗

bioRxiv2024

Cellular Function of a Biomolecular Condensate Is Determined by Its Ultrastructure

Biomolecular condensates play key roles in the spatiotemporal regulation of cellular processes. Yet, the relationship between atomic features and condensate function remains poorly understood. We studied this relationship using the polar organizing protein Z (PopZ) as a model system, revealing how its material properties and cellular function depend on its ultrastructure. We revealed PopZs hierarchical assembly into a filamentous condensate by integrating cryo-electron tomography, biochemistry, single-molecule techniques, and molecular dynamics simulations. The helical domain drives filamentation and condensation, while the disordered domain inhibits them. Phase-dependent conformational changes prevent interfilament contacts in the dilute phase and expose client binding sites in the dense phase. These findings establish a multiscale framework that links molecular interactions and condensate ultrastructure to macroscopic material properties that drive cellular function.

cell biology↗

bioRxiv2023

Probing different conformational states of polar organizing protein Z with AlphaFold

AlphaFold has been a remarkably powerful tool to predict the structures of proteins. Here, we demonstrate that AlphaFold is capable of sampling different conformational states of the oligomerization domain of polar organizing protein Z (PopZ). More broadly, our work suggests that conformational heterogeneity can be extracted from AlphaFold without directly modifying the input multiple sequence alignment.

biophysics↗

bioRxiv2023

Aberrant phase separation is a common killing strategy of positively charged peptides in biology and human disease

Positively charged repeat peptides are emerging as key players in neurodegenerative diseases. These peptides can perturb diverse cellular pathways but a unifying framework for how such promiscuous toxicity arises has remained elusive. We used mass-spectrometry-based proteomics to define the protein targets of these neurotoxic peptides and found that they all share similar sequence features that drive their aberrant condensation with these positively charged peptides. We trained a machine learning algorithm to detect such sequence features and unexpectedly discovered that this mode of toxicity is not limited to human repeat expansion disorders but has evolved countless times across the tree of life in the form of cationic antimicrobial and venom peptides. We demonstrate that an excess in positive charge is necessary and sufficient for this killer activity, which we name polycation poisoning. These findings reveal an ancient and conserved mechanism and inform ways to leverage its design rules for new generations of bioactive peptides.

cell biology↗

bioRxiv2022

Generative modeling of short, disordered proteins with homogeneous sequence composition

Protein design has seen remarkable progress in the past decade, with numerous examples of de novo proteins with novel topologies and functions being successfully synthesized. Computational tools have played a large role in the ability to rationally design proteins. Recently, there have been numerous successes applying deep learning techniques to protein design that have demonstrated comparable or significantly improved performance over traditional energy-based approaches. However, the protein design problem has been less well explored for disordered proteins or, more generally, proteins with conformational heterogeneity. In this work, we demonstrate that if one approximates the spatial output of a coarse-grained molecular dynamics simulation as a multivariate normal distribution parameterized by a mean vector (representing an ensemble-averaged pairwise distance map) and covariance matrix, one can train a generative model to learn the distribution of these parameters across a set of sequences. Specifically, we encoded the mean vector and covariance matrix for each sequence in a low-dimensional space via a fixed linear transformation and trained a masked auto-encoder to accurately learn the distribution of this low-dimensional output. Furthermore, by sampling from the masked auto-encoder and transforming the generated samples back into their original high-dimensional space, one can generate realistic, ensemble-averaged pairwise distance maps. These results were demonstrated on coarse-grained simulation data derived from approximately 2000 distinct sequences, each sequence being 24 residues in length and consisting exclusively of glycine, serine, glutamate, and lysine. Though this set of sequences is relatively homogeneous in composition, we speculate our approach can be applied to disordered sequences of longer length and more heterogeneous composition, given the appropriate training set.

bioinformatics↗

bioRxiv2021

Poly(A)-binding protein is an ataxin-2 chaperone that emulsifies biomolecular condensates.

Biomolecular condensation underlies the biogenesis of an expanding array of membraneless assemblies, including stress granules (SGs) which form under a variety of cellular stresses. Advances have been made in understanding the molecular grammar that dictates the behavior of a few key scaffold proteins that make up these phases but how the partitioning of hundreds of other SG proteins is regulated remains largely unresolved. While investigating the rules that govern the condensation of ataxin-2, a SG protein implicated in neurodegenerative disease, we unexpectedly identified a short 14aa sequence that acts as an ataxin-2 condensation switch and is conserved across the eukaryote lineage. We identify poly(A)-binding proteins as unconventional RNA-dependent chaperones that control this regulatory switch. Our results uncover a hierarchy of cis and trans interactions that fine-tune ataxin-2 condensation and reveal a new molecular function for ancient poly(A)-binding proteins as emulsifiers of biomolecular condensate proteins. These findings may inspire novel approaches to therapeutically target aberrant phases in disease.

cell biology↗

bioRxiv2021

A modular platform for engineering function ofnatural and synthetic biomolecular condensates

Phase separation is emerging as a universal principle for how cells use dynamic subcompartmentalization to organize biochemical reactions in time and space1,2. Yet, whether the emergent physical properties of these biomolecular condensates are important for their biological function remains unclear. The intrinsically disordered protein PopZ forms membraneless condensates at the poles of the bacterium Caulobacter crescentus and selectively sequesters kinase-signaling cascades to regulate asymmetric cell division3-5. By dissecting the molecular grammar underlying PopZ phase separation, we find that unlike many eukaryotic examples, where unstructured regions drive condensation6,7, a structured domain of PopZ drives condensation, while conserved repulsive features of the disordered region modulate material properties. By generating rationally designed PopZ mutants, we find that the exact material properties of PopZ condensates directly determine cellular fitness, providing direct evidence for the physiological importance of the emergent properties of biomolecular condensates. Our work codifies a clear set of design principles illuminating how sequence variation in a disordered domain alters the function of a widely conserved bacterial condensate. We used these insights to repurpose PopZ as a modular platform for generating synthetic condensates of tunable function in human cells.

cell biology↗

bioRxiv2021

ATAC-seq reveals megabase-scale domains of a bacterial nucleoid

Here we adapted ATAC-seq to probe chromosome accessibility of bacterial cells. We found that the chromosome of Caulobacter crescentus is composed of eight differentially compacted regions we name Chromosomal Accessibility Domains (CADs). This domain structure is depended on the cell cycle stage, DNA gyrase activity, and the nucleoid-associated protein (NAP) GapR, but not on the function of SMC. We show the chromosome is punctuated by four highly transposase-inaccessible transcribed regions (HINTs). The HINTs include Caulobacters ribosomal RNA clusters and its largest ribosomal protein gene cluster. Further, we show that HINTs are also formed by rDNA in E. coli and provide evidence that their high levels of transcription do not strictly govern their formation. Overall, this work argues that physical forces, including those created by the activities of DNA gyrase and specific NAPs, significantly contribute to bacterial nucleoid structure at the megabase scale. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/426053v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@2b9812org.highwire.dtl.DTLVardef@cd430org.highwire.dtl.DTLVardef@3ed5cforg.highwire.dtl.DTLVardef@1471dad_HPS_FORMAT_FIGEXP M_FIG C_FIG SignificanceIn bacteria, chromosomal DNA is highly compacted and organized. Many forces contribute to bacterial DNA compaction, including the transcription, DNA replication, and the activities of topoisomerases and nucleoid-associated proteins. At the megabase scale, the resulting chromosome structure is important for coordinating cell cycle events; for example, in E. coli the improper structuring of a Mb-scale nucleoid domain leads to errors in the fidelity of chromosome segregation. It was previously unknown whether Mb-scale regions of a bacterial chromosome could be differentially compacted, and which factors might contribute to this spatial variation in compaction. Our work provides a novel method for measuring global chromosome compaction in bacteria. By applying this method in the bacterium Caulobacter crescentus, we show that the Caulobacter nucleoids compaction varies between megabase-scale domains. We also show that DNA gyrase and the nucleoid-associated protein GapR are key players in shaping the uneven compaction of the chromosome.

microbiology↗

bioRxiv2019

The in situ structure of Parkinson’s disease-linked LRRK2

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinsons disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using in situ cryo-electron tomography and subtomogram averaging, we reveal a 14-[A] structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double-helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase points towards the microtubule, while the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to obtain structures of previously unsolved proteins in their cellular environment and provides insights into LRRK2 function and pathogenicity.

biophysics↗

bioRxiv2017

Phospho-signal flow from a pole-localized microdomain spatially patterns transcription factor activity

Selective recruitment and concentration of signaling proteins within membrane-less compartments is a ubiquitous mechanism for subcellular organization. However, little is known about how such dynamic recruitment patterns intracellular signaling and cellular development. Here, we combined transcriptional profiling, reaction-diffusion modeling, and single-molecule tracking to study signal exchange in and out of a microdomain at the cell pole of the asymmetrically dividing bacterium Caulobacter crescentus. Our study revealed that the microdomain is selectively permeable, and that each protein in the signaling pathway that activates the cell fate transcription factor CtrA is sequestered and uniformly concentrated within the microdomain or its proximal membrane. Restricted rates of entry into and escape from the microdomain enhance phospho-signaling, leading to a sublinear gradient of CtrA~P along the long axis of the cell. The spatial patterning of CtrA~P creates a gradient of transcriptional activation that serves to prime asymmetric development of the two daughter cells.

cell biology↗