Biology subjects

Hubrich, J.

Publications and source records attributed to Hubrich, J..

5 recordsLinked to original sources

bioRxiv2026

Paralemmin-2 is a membrane-anchored cytoskeletal constituent of Axon Initial Segments and nodes of Ranvier

The axon initial segment (AIS) and nodes of Ranvier (NoR) are essential for action potential initiation and propagation. They share many features of their molecular architecture, and their assembly mechanisms converge on the membrane-associated periodic skeleton (MPS). Here, we identify Paralemmin-2 (Palm2) as a component of both the AIS and NoR. Palm2 depletion shortens the AIS and reduces neuronal excitability. Endogenous Palm2 in the AIS is non-periodic, but upon overexpression it associates with the MPS, localizing to actin rings and reducing {beta}IV-spectrin abundance and periodicity. In NoR of the central and peripheral nervous system, Palm2 localizes to different subdomains - nodal or paranodal, respectively. Palm2, and its homolog Palm1, bind the deubiquitinase USP7, implicating paralemmins in proteostasis at the MPS. The complementary localizations of Palm2 and Palm1 at the AIS/NoR or axon shafts, respectively, parallel the distributions of {beta}-spectrin and ankyrin isoforms between these axonal compartments. We propose that Palm2 modulates the submembrane cytoskeleton and its membrane attachment, and thus contributes to the assembly, functioning and remodeling of the AIS and NoR.

cell biology↗

bioRxiv2024

Paralemmin-1 controls the nanoarchitecture of the neuronal submembrane cytoskeleton

The Membrane-associated Periodic Skeleton (MPS) is a specialized submembrane cytoskeleton of neuronal cells, characterized by a highly ordered 190 nm periodic lattice, with emerging functions in mechanical resilience, inter- and intracellular signaling, and action potential transmission. Here, we identify Paralemmin-1 (Palm1) as a new component and regulator of the MPS. Palm1 binds to the N-terminal region of {beta}II-spectrin, a core MPS component, and is periodically organized along the axon in hippocampal neurons. Applying the 3D imaging power of MINFLUX, we locate Palm1 in close proximity (<20 nm) to the actin-capping protein and MPS component adducin. Functionally, Palm1 overexpression enhances the degree of periodicity of several MPS proteins ({beta}II-spectrin, adducin, and ankyrinB) without altering their local concentrations, while the knock-out severely compromises the MPS structure and modifies electrophysiological properties of neurons. Both the MPS-binding and remodelling activities of Palm1 are abolished by mutating a single amino acid (W54A) in the conserved Paralemmin sequence motif. Our findings identify Palm1 as the first protein specifically dedicated to organizing the MPS, and will advance the understanding of the regulation of MPS assembly and remodelling, as well as of the Paralemmin protein family.

cell biology↗

bioRxiv2024

MINFLUX Reveals Dynein Stepping in Live Neurons

Dynein is the primary molecular motor responsible for retrograde intracellular transport of a variety of cargoes, performing successive nanometer-sized steps within milliseconds. Due to the limited spatiotemporal precision of established methods for molecular tracking, current knowledge of dynein stepping is essentially limited to slowed-down measurements in vitro. Here, we use MINFLUX fluorophore localization to directly track CRISPR/Cas9-tagged endogenous dynein with nanometer/millisecond precision in living primary neurons. We show that endogenous dynein primarily takes 8 nm steps, including frequent sideways steps but few backward steps. Strikingly, the majority of direction reversals between retrograde and anterograde movement occurred on the time scale of single steps (16 ms), suggesting a rapid regulatory reversal mechanism. Tug-of-war-like behavior during pauses or reversals was unexpectedly rare. By analyzing the dwell time between steps, we concluded that a single rate-limiting process underlies the dynein stepping mechanism whereby dynein consumes one adenosine 5-triphosphate (ATP) per step. Our study underscores the power of MINFLUX localization to elucidate the spatiotemporal changes underlying protein function in living cells.

biophysics↗

bioRxiv2023

Neuronal activity modulates the incorporation of newly translated PSD-95 into a robust structure as revealed by STED and MINFLUX

The postsynaptic density component PSD-95 undergoes activity-dependent plasticity mechanisms that rely on protein synthesis and structural remodeling. How synaptic activity can influence these dynamics at the single synapse level remains unclear. Here we combine genome-editing, pulse-chase experiments, STED and 3D MINFLUX nanoscopy on hippocampal neuronal cultures to study the integration of newly translated PSD-95 molecules at postsynaptic sites and their rearrangement within individual clusters at near-molecular resolution. We show that the amount of newly translated PSD-95 recruited to individual synapses scales with synaptic size, and modulates in a bidirectional manner, resulting in less new protein following excitatory and more new protein following inhibitory stimulation. Furthermore, we show that within synaptic clusters PSD-95 has a dispersed organization that is largely robust to long-lasting changes in activity. Altogether, this work sheds new light on the mechanisms underlying plasticity at the single synapse level, adding previously inaccessible information.

neuroscience↗

bioRxiv2022

Synaptic status differentially regulates neurofilaments in dendritic spines

Neurofilaments are one of the main cytoskeletal components in neurons and they can be found in the form of oligomers at pre- and postsynapses. How their presence is regulated at the postsynapse remains widely unclear. Here we systematically quantified by immunolabeling the occurrence of the neurofilament isoform triplet neurofilament light (NFL), medium (NFM), and heavy (NFH) at the postsynapse with STED nanoscopy together with markers of synaptic strength and activity. Our data shows that within dendritic spines neurofilament isoforms rarely colocalize with each other and that they are present to different extents, with NFL being the most abundant isoform. The amount of the three isoforms correlates with markers of postsynaptic strength and presynaptic activity to varying degrees: while NFL shows moderate correlation to both synaptic traits suggesting its involvement in synaptic response, NFM and NFH were only correlated on a low level. By quantifying the presence of neurofilaments at the postsynapse within the context of the synaptic status, this work sheds new light on the regulation of synaptic neurofilaments and their possible contribution to psychiatric disorders.

neuroscience↗