Biology subjects

Rout, M.

Publications and source records attributed to Rout, M..

5 recordsLinked to original sources

bioRxiv2024

Development and Characterization of 50 nanometer diameter Genetically Encoded Multimeric Nanoparticles

The mechanisms that regulate the physical properties of the cell interior remain poorly understood, especially at the mesoscale. Many crucial macromolecules and molecular assemblies such as ribosomes, RNA polymerase, and biomolecular condensates span the mesoscale size range, and changes in mesoscale physical properties have been suggested to be crucial for both normal physiology and disease. Therefore, we need better tools to study the cellular environment at this scale. Physical properties of the cell can be inferred through analysis of the motion of tracer nanoparticles, an approach called nanorheology. This requires the introduction of nanoparticles to cells, which can be labor intensive and slow. Genetically Encoded Multimeric nanoparticles (GEMs) were recently developed to address this limitation. GEMs consist of scaffold proteins fused to fluorescent tags that self-assemble into bright and stable nanoparticles of defined geometry. However, extremely sensitive microscopes were required to track previous (40nm-GEMs). Here, we describe the development and characterization of 50 nm diameter GEMs (50nm-GEMs) that are brighter and probe a slightly longer length scale. 50nm-GEMs will make high-throughput nanorheology accessible to a broader range of researchers and reveal new insights into the biophysical properties of cells.

biophysics↗

bioRxiv2024

Characterizing interactions in the nuclear pore complex transporter using novel site-specific deuteration and SANS

We describe an unprecedented SANS/solvent matched experiment with the aim of providing a meso-scale dynamic structural description of fuzzy complexes such as those formed by nuclear transport factors as they ferry cargo across the nuclear pore complex, whose description is normally limited by the large number of states and rapid time scales of interconversion. These contain repeat short linear interaction domains which are common features of many intrinsically disordered functional proteins - e.g., transcriptional regulators, and RNA-interaction domains associated with liquid-liquid phase separated non-membranous organelles. The novel approach uses site-specific deuteration, SANS, and model fitting to provide changes to average spatial distributions between interacting domains of FG Nups upon binding to the nuclear transport factor NTF2. The results support the fully disordered nature of phenylalanyl-glycyl repeats within FG Nups in their interactions with the nuclear transport factor in vitro, as well as the absence of significant inter-aromatic contacts, or of interchain linkage in complexes.

biophysics↗

bioRxiv2024

Exportin-1 functions as an adaptor for transcription factor-mediated docking of chromatin at the nuclear pore complex.

Nuclear pore proteins (Nups) in yeast, flies and mammals physically interact with hundreds or thousands of chromosomal sites, which impacts transcriptional regulation. In budding yeast, transcription factors mediate interaction of Nups with enhancers of highly active genes. To define the molecular basis of this mechanism, we exploited a separation-of-function mutation in the Gcn4 transcription factor that blocks its interaction with the nuclear pore complex (NPC) without altering its DNA binding or activation domains. SILAC mass spectrometry revealed that this mutation reduces the interaction of Gcn4 with the highly conserved nuclear export factor Crm1/Xpo1. Crm1 both interacts with the same sites as Nups genome-wide and is required for Nup2 to interact with the yeast genome. In vivo, Crm1 undergoes extensive and stable interactions with the NPC. In vitro, Crm1 binds to Gcn4 and these proteins form a complex with the nuclear pore protein Nup2. Importantly, the interaction between Crm1 and Gcn4 does not require Ran-GTP, suggesting that it is not through the nuclear export sequence binding site. Finally, Crm1 stimulates DNA binding by Gcn4, supporting a model in which allosteric coupling between Crm1 binding and DNA binding permits docking of transcription factor-bound enhancers at the NPC.

cell biology↗

bioRxiv2022

Cryomilling Tethered Chromatin Conformation Capture reveal new insights into inter-chromosomal interactions

Traditional methods used to map the three-dimensional organization of chromatin in-situ generally involve chromatin conformation capture by formaldehyde crosslinking, followed by detergent solubilization and enzymatic digestion of DNA. Ligation of proximal DNA fragments followed by next generation sequencing (NGS) generates contact information that enables a global view of the chromatin conformation. Here, we explore the use of cryomilling to physically fragmentize the cells under cryogenic conditions to probe chromatin interactions in the cryomilled cell fragments by the tethered chromatin conformation capture (TCC). Our results show that cryomilling TCC (CTCC) can generate a global contact map similar to that obtained with in-situ Hi-C. This result suggests that summation of chromatin interactions mapped in individual subcellular fragments can reconstitute the global contact map of intact cells in an ensemble manner, paving the way for chromatin conformation analyses of solid tissue by CTCC. Compared with the conventional in-situ methods such as Hi-C, CTCC shows more uniform access to different subcompartments of the folded genome. On the other hand, most inter-chromosomal (trans) contacts are diminished or lost in CTCC except for a group of unique trans contacts that remain intact throughout the cryomilling and in- vitro crosslinking steps. These apparently ultra-stable trans interactions have much enhanced signal in CTCC due to the elimination of signals of most, presumably weak and transient trans interactions. Systematic and comparative analyses between CTCC and in-situ Hi-C provide further insights into the chromatin structure organization and reveal a generally unentangled chromosome interface and the existence of stable inter-chromosomal contacts that may represent intermingled inter-chromosomal interfaces.

genomics↗

bioRxiv2021

Comprehensive Structure and Functional Adaptations of the Yeast Nuclear Pore Complex

Nuclear Pore Complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the yeast NPC in which the inner ring is resolved by cryo-EM at - helical resolution to show how flexible connectors tie together different structural and functional layers in the spoke. These connectors are targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and karyopherins have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We also provide evidence for three major NPC variants that foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies to provide a comprehensive model of the in situ NPC with a radially-expanded inner ring. Our model reveals novel features of the central transporter and nuclear basket, suggests a role for the lumenal ring in restricting dilation and highlights the structural plasticity required for transport by the NPC.

biophysics↗