Biology subjects

Peukes, J.

Publications and source records attributed to Peukes, J..

8 recordsLinked to original sources

bioRxiv2026

The Molecular Interplay of Tau and Motor Proteins on Microtubules

Tau is a microtubule (MT)-associated protein essential for neuronal cytoskeletal organization and intracellular transport, yet how tau physically regulates motor proteins remains elusive. Here, we integrate single-molecule imaging, cryo-electron microscopy, and molecular dynamics (MD) simulations to investigate the molecular mechanisms underlying tau-mediated inhibition of kinesin-1 and cytoplasmic dynein motility. We show that tau suppresses MT landing and triggers frequent pausing and premature detachment when motors encounter tau-enriched patches. Unexpectedly, tau impairs each motor using different mechanisms. Tau and kinesin-1 compete for the same surface on tubulin, precluding simultaneous binding to the same MT site. On the other hand, while dynein and tau can co-occupy the same tubulin, tau inhibits the transition of dynein into its high-affinity MT-binding state. Thus, tau can act as a physical or a conformational roadblock in a concentration-dependent manner, exerting distinct structural constraints on kinesin and dynein transport. These findings establish a structural framework for tau-mediated regulation of axonal transport.

biophysics↗

bioRxiv2026

Microtubule Engagement by the WD40-Containing Tail of Kinesin-4 KIF21B

Kinesin tails are structurally diverse and mediate a range of functions, including autoinhibition, cargo binding, and microtubule regulation. Within the kinesin-4 family, KIF21A and KIF21B have emerged as key regulators of microtubule network organization and dynamics in neurons and immune cells, yet the molecular basis of this activity has remained unclear. Here, we combined single-particle cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) to examine how the KIF21B tail engages microtubules. We find that conserved residues in the WD40 {beta}-propeller and an adjacent N-terminal linker contact successive tubulin dimers along a single protofilament, forming an extended longitudinal binding mode that spans both the intradimer and interdimer interfaces. Cryo-EM 3D classification further revealed two distinct engagement states, a tilted state, in which the {beta}-propeller makes partial contacts with the microtubule while the linker remains anchored, and a flat state, in which the {beta}-propeller lies flush with the lattice surface. Cryo-ET of full-length KIF21B reveals multiple binding configurations on the microtubule lattice, including orientations consistent with crosslinking adjacent microtubules. Together, these findings provide a structural framework for tail-mediated microtubule attachment by a neuronal kinesin-4 and suggest how its distinctive WD40 domain may contribute to microtubule regulation and organization of microtubule arrays.

biochemistry↗

bioRxiv2026

High Accuracy Fluorescence Guided Focused Ion Beam Milling

Cryo-electron tomography (cryo-ET) is a powerful approach for visualizing macromolecular structures directly within cells, but its broader application is limited by the difficulty of reliably targeting specific structures during sample preparation. In particular, capturing small or rare objects within cryo-focused ion beam (cryo-FIB) milled lamellae remains a major bottleneck. Here, we present two fluorescence-guided cryo-FIB milling workflows that overcome key sources of targeting error and enable routine capture of structures across a broad range of spatial scales. For larger targets (>500 nm), we present an improved registration-based strategy combining FIB-milled fiducials with depth correction for refractive index mismatch. For smaller targets (150-500 nm), we implement real-time fluorescence guided milling on a commercially available cryo-FIB-SEM platform enabling consistent recovery of small, low-copy organelles such as centrosomes and primary cilia. Both workflows are implemented in a new open-source package fibsemOS and together expand the range of cellular structures accessible to cryo-ET and establish a broadly deployable framework for targeted in situ structural biology.

cell biology↗

bioRxiv2025

AreTomoLive: Automated reconstruction of comprehensively-corrected and denoised cryo-electron tomograms in real-time and at high throughput

A high throughput processing pipeline that performs comprehensive corrections is needed to realize the full potential of cryo-electron tomography and subtomogram averaging. The fields fragmented software landscape remains a significant hurdle to this end. Here we present AreTomoLive, an automated real-time pipeline composed of two GPU-accelerated packages. The first, AreTomo3, streamlines tomographic alignment and reconstruction, with new features to fully account for sample geometry, locally correct the contrast transfer function, and curate data for downstream tasks. The second package, DenoisET, is a new implementation of the machine learning algorithm Noise2Noise and runs in parallel with AreTomo3 to perform contrast enhancement. To reduce barriers to routine use, AreTomoLive prioritizes automation: AreTomo3 autonomously pauses and reactivates processing depending on the status of data collection, while DenoisET algorithmically determines when to transition from training to inference. AreTomoLive endeavors to advance cryoET for in situ structural analysis with its comprehensive corrections and full automation.

cell biology↗

bioRxiv2024

A physical model for M1-mediated influenza A virus assembly

Influenza A virus particles assemble at the plasma membrane of infected cells. During assembly all components of the virus come together in a coordinated manner to deform the membrane into a protrusion eventually forming a new, membrane-enveloped virus. Here we integrate recent molecular insights of this process, particularly concerning the structure of the matrix protein 1 (M1), within a theoretical framework describing the mechanics of virus assembly. Our model describes M1 polymerization and membrane protrusion formation, explaining why it is efficient for M1 to form long strands assembling into helices in filamentous virions. Eventually, we find how the architecture of M1 helices is controlled by physical properties of viral proteins and the host cell membrane. Finally, by considering the growth force and speed of viral filaments, we propose that the helical geometry of M1 strands might have evolved to optimize for fast and efficient virus assembly and growth. SignificanceInfluenza A virus remains a major threat to public health. Its most abundant viral protein, matrix protein 1 (M1), forms an endoskeleton underneath the viral membrane, but how this endoskeleton contributes to the virus lifecycle is poorly understood. Combining cryo-electron tomography data and structural data with theoretical predictions, we explain how the energetically favorable polymerization of M1 into helical strands mediates the membrane deformations that permit the virus to exit infected cells. Our analysis of M1s variable architecture provides insights into adaptive strategies of the virus for efficient growth under variable local conditions. The quantitative framework developed in this study could be extrapolated to other enveloped viruses and generally applied to protein-driven membrane deformations.

biophysics↗

bioRxiv2021

The molecular infrastructure of glutamatergic synapses in the mammalian forebrain

Glutamatergic synapses form the vast majority of connections within neuronal circuits, but how these subcellular structures are molecularly organized within the mammalian brain is poorly understood. Conventional electron microscopy using chemically fixed, metal- stained tissue has identified a proteinaceous, membrane-associated thickening called the postsynaptic density (PSD). Here, we combined mouse genetics and cryo-electron tomography to determine the 3D molecular architecture of fresh isolated and anatomically intact synapses in the adult forebrain. The native glutamatergic synapse did not consistently show a higher density of proteins at the postsynaptic membrane thought to be characteristic of the PSD. Instead, a synaptoplasm consisting of cytoskeletal elements, macromolecular complexes and membrane-bound organelles extended throughout the pre- and post- synaptic compartments. Snapshots of active processes gave insights into membrane remodeling processes. Clusters of up to 60 ionotropic glutamate receptors were positioned inside and outside the synaptic cleft. Together, these information-rich tomographic maps present a detailed molecular framework for the coordinated activity of synapses in the adult mammalian brain.

neuroscience↗

bioRxiv2020

Structures, conformations and distributions of SARS-CoV-2 spike protein trimers on intact virions

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind the ACE2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates the exposure of its receptor binding site and later undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy. The structure and distribution of S on the virion surface, however, has not been characterised. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions, determining the high-resolution structure, conformational flexibility and distributions of S trimers in situ on the virion surface. These results provide a basis for understanding the conformations of S present on the virion, and for studying their interactions with neutralizing antibodies.

microbiology↗

bioRxiv2020

The native structure of the full-length, assembled influenza A virus matrix protein, M1.

Influenza A virus causes millions of severe illnesses during annual epidemics. The most abundant protein in influenza virions is the matrix protein M1 that mediates virus assembly by forming an endoskeleton beneath the virus membrane. The structure of full-length M1, and how it oligomerizes to mediate assembly of virions, is unknown. Here we have determined the complete structure of assembled M1 within intact virus particles, as well as the structure of M1 oligomers reconstituted in vitro. We found that the C-terminal domain of M1 is disordered in solution, but can fold and bind in trans to the N-terminal domain of another M1 monomer, thus polymerising M1 into linear strands which coat the interior surface of the assembling virion membrane. In the M1 polymer, five histidine residues, contributed by three different M1 monomers, form a cluster that can serve as the pH-sensitive disassembly switch after entry into a target cell. These structures therefore provide mechanisms for influenza virus assembly and disassembly.

biochemistry↗