Biology subjects

Mahamid, J.

Publications and source records attributed to Mahamid, J..

40 records · Page 3Linked to original sources

bioRxiv2020

In-cell architecture of an actively transcribing-translating expressome

Structural biology performed inside cells can capture molecular machines in action within their native context. Here we develop an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combine whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpoint NusA at the interface between a NusG-bound elongating RNAP and the ribosome, and propose it could mediate transcription-translation coupling. Translation inhibition dissociates the expressome, whereas transcription inhibition stalls and rearranges it, demonstrating that the active expressome architecture requires both translation and transcription elongation within the cell.

molecular biology↗

bioRxiv2020

In cell architecture of the nuclear pore complex and snapshots of its turnover

Nuclear pore complexes (NPCs) mediate exchange across the nuclear envelope. They consist of hundreds of proteins called nucleoporins (Nups) that assemble in multiple copies to fuse the inner and outer nuclear membranes. Elucidating the molecular function and architecture of NPCs imposes a formidable challenge and requires the convergence of in vitro and in situ approaches. How exactly NPC architecture accommodates processes such as mRNA export or NPC assembly and turnover inside of cells remains poorly understood. Here we combine integrated in situ structural biology, correlative light and electron microscopy with yeast genetics to structurally analyze NPCs within the native context of Saccharomyces cerevisiae cells under conditions of starvation and exponential growth. We find an unanticipated in situ layout of nucleoporins with respect to overall dimensions and conformation of the NPC scaffold that could not have been predicted from previous in vitro analysis. Particularly striking is the configuration of the Nup159 complex, which appears critical to spatially accommodate not only mRNA export but also NPC turnover by selective autophagy. We capture structural snapshots of NPC turnover, revealing that it occurs through nuclear envelope herniae and NPC-containing nuclear vesicles. Our study provides the basis for understanding the various membrane remodeling events that happen at the interface of the nuclear envelope with the autophagy apparatus and emphasizes the need of investigating macromolecular complexes in their cellular context.

biophysics↗

bioRxiv2019

Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies

Spatially-controlled cell adhesion on electron microscopy (EM) supports remains a bottleneck in specimen preparation for cellular cryo-electron tomography. Here, we describe contactless and mask-free photo-micropatterning of EM grids for site-specific deposition of extracellular matrix-related proteins. We attained refined cell positioning for micromachining by cryo-focused ion beam milling. Complex patterns generated predictable intracellular organization, allowing direct correlation between cell architecture and in-cell 3D-structural characterization of the underlying molecular machinery.

cell biology↗

bioRxiv2016

The centrosome is a selective phase that nucleates microtubules by concentrating tubulin

Centrosomes are non-membrane-bound compartments that nucleate microtubule arrays. They consist of nanometer-scale centrioles surrounded by a micron-scale, dynamic assembly of protein called the pericentriolar material (PCM). To study how PCM forms a spherical compartment that nucleates microtubules, we reconstituted PCM-dependent microtubule nucleation in vitro using recombinant C.elegans proteins. We found that macromolecular crowding drives phase separation of the key PCM scaffold protein SPD-5 into spherical droplets that morphologically and dynamically resemble in vivo PCM. These SPD-5 droplets recruited the microtubule polymerase ZYG-9 (XMAP215 homologue) and the microtubule-stabilizing protein TPXL-1 (TPX2 homologue). Together, these three proteins concentrated tubulin ~4- fold over background, which was sufficient to reconstitute nucleation of microtubule asters in vitro. Our results suggest that in vivo PCM is a selective phase that organizes microtubule arrays through localized concentration of tubulin by microtubule effector proteins.\n\nOne Sentence SummaryPhase separation of C. elegans centrosome proteins drive the formation of micron-sized microtubule organizing centers.

cell biology↗