Biology subjects

Young, L. N.

Publications and source records attributed to Young, L. N..

3 recordsLinked to original sources

bioRxiv2024

ExoSloNano: Multi-Modal Nanogold Tags for identi-fication of Macromolecules in Live Cells & Cryo-Electron Tomograms

In situ cryo-Electron Microscopy (cryo-EM) enables the direct interrogation of structure-function relationships by resolving macromolecular structures in their native cellular environment. Tremendous progress in sample preparation, imaging and data processing over the past decade has contributed to the identification and determination of large biomolecular complexes. However, the majority of proteins are of a size that still eludes identification in cellular cryo-EM data, and most proteins exist in low copy numbers. Therefore, novel tools are needed for cryo-EM to identify the vast majority of macromolecules across multiple size scales (from microns to nanometers). Here, we introduce and validate novel nanogold probes that enable the detection of specific proteins using cryo-ET (cryo-Electron Tomography) and resin-embedded correlated light and electron microscopy (CLEM). We demonstrate that these nanogold probes can be introduced into live cells, in a manner that preserves intact molecular networks and cell viability. We use this system to identify both cytoplasmic and nuclear proteins by room temperature EM, and resolve associated structures by cryo-ET. We further employ gold particles of different sizes to enable future multiplexed labeling and structural analysis. By providing high efficiency protein labeling in live cells and molecular specificity within cryo-ET tomograms, we establish a broadly enabling tool that significantly expands the proteome available to electron microscopy.

biophysics↗

bioRxiv2019

ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer

The autophagy-initiating human ULK complex consists of the kinase ULK1/2, FIP200, ATG13, and ATG101. Hydrogen-deuterium exchange mass spectrometry was used to map their mutual interactions. The N-terminal 640 residues (NTD) of FIP200 interact with the C-terminal IDR of ATG13. Mutations in these regions abolish their interaction. Negative stain electron microscopy (EM) and multiangle light scattering showed that FIP200 is a dimer whilst a single molecule each of the other subunits is present. The FIP200 NTD is flexible in the absence of ATG13, but in its presence adopts the shape of the letter C ~20 nm across. The ULK1 EAT domain interacts loosely with the NTD dimer, while the ATG13-ATG101 HORMA dimer does not contact the NTD. Cryo-EM of the NTD dimer revealed a structure similarity to the scaffold domain of TBK1, suggesting an evolutionary similarity between the autophagy initiating TBK1 kinase and the ULK1 kinase complex. SummaryThe human ULK complex consists of ULK1/2, FIP200, ATG13, and ATG101. We found that the FIP200 N-terminal domain is a C-shaped dimer that binds directly to a single ATG13 molecule and serves as the organizing hub of the complex.

biochemistry↗

bioRxiv2019

Structural Pathway for Allosteric Activation of the Autophagic PI 3-Kinase Complex I

Autophagy induction by starvation and stress involves the enzymatic activation of the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1). The inactive basal state of PI3KC3-C1 is maintained by inhibitory contacts between the VPS15 protein kinase and VPS34 lipid kinase domains that restrict the conformation of the VPS34 activation loop. Here, the pro-autophagic MIT domain-containing protein NRBF2 was used to map the structural changes leading to activation. Cryo-EM was used to visualize stepwise PI3KC3-C1 activating effects of binding the NRFB2 MIT domains. Binding of a single NRBF2 MIT domain to bends the helical solenoid of the VPS15 scaffold, displaces the protein kinase domain of VPS15, and releases the VPS34 kinase domain from the inhibited conformation. Binding of a second MIT stabilizes the VPS34 lipid kinase domain in an active conformation that has an unrestricted activation loop and is poised for access to membranes.

biochemistry↗