Biology subjects

Kapadia, A. B.

Publications and source records attributed to Kapadia, A. B..

4 recordsLinked to original sources

bioRxiv2026

Protocol for studying membrane protein dynamics and associated synaptic vesicle recruitment on native membrane sheets

Plasma membrane sheets generated by controlled mechanical disruption provide direct access to the cytosolic face of the plasma membrane while preserving the native organization of membrane-associated proteins and lipids. Here, we present a protocol for generating and validating sonication-derived plasma membrane sheets from cultured cells, primary neurons, and isolated synaptosomes. We further describe their application for live and fixed imaging of membrane protein localization, organization, conformational dynamics, and protein-protein interactions, as well as quantitative membrane-associated synaptic vesicle recruitment assays. This versatile platform preserves the native membrane environment while enabling direct visualization and quantitative analysis of membrane-associated processes at high spatial resolution. The protocol can be readily adapted to investigate diverse membrane proteins, lipid-dependent mechanisms, and vesicle tethering events across a wide range of cellular systems. HighlightsO_LIPreparation of plasma membrane sheets exposing the cytosolic membrane surface C_LIO_LIOptimization and validation of membrane sheets from cultured cells, neurons, and isolated synaptosomes C_LIO_LIAdaptable platform for studying membrane protein interactions in native membranes C_LIO_LIQuantitative assay for membrane-associated synaptic vesicle recruitment C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/736009v1_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@e725a0org.highwire.dtl.DTLVardef@1576963org.highwire.dtl.DTLVardef@1c7ea73org.highwire.dtl.DTLVardef@1f13e94_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

APP-CTFβ/C99 oligomers drive synaptic vesicle tethering through C-terminal interactions

Proteolytic processing of the amyloid precursor protein (APP) generates a 99-amino acid precursor, {beta}-carboxyl-terminal fragments (APP-CTF{beta} or C99). Upon {gamma}-secretase inhibition, APP-CTF{beta} accumulates and induces synaptic defects, resulting in neuronal hyperactivity. However, mechanistic insights in the critical role of APP-CTF{beta} has not been completely elucidated. Here, we show that in primary neurons expressing human APP-CTF{beta} (C99) variants, acute {gamma}-secretase inhibition selectively increases evoked synaptic vesicle release in cells, whereas deletion of the C-terminus abolishes this effect. Using single-molecule approaches and reconstituted membrane systems, we demonstrate that accumulation of APP-CTF{beta} promotes its oligomerisation. In particular, APP-CTF{beta} oligomers augment synaptic vesicle tethering via their C-terminal domain. This effect is driven by the interaction with synaptic vesicle proteins, independent of the YENPTY binding motif. Additionally, APP-CTF{beta} oligomers were associated with alterations in membrane lipid organization. Together, our findings identify APP-CTF{beta} oligomerization as a constitutional gain-of-function mechanism that enhances presynaptic vesicle tethering and release, providing mechanistic insight into how altered APP processing regulates synaptic activity. Short summary{gamma}-Secretase-dependent accumulation of APP-CTF{beta} transforms a transient APP processing intermediate into a membrane-associated oligomeric scaffold that promotes synaptic vesicle tethering enhancing neurotransmitter release. HighlightsO_LIAPP-CTF{beta} self-assembles into higher-order oligomeric assemblies C_LIO_LIAPP-CTF{beta} oligomers promote synaptic vesicle tethering via their C-terminal domain C_LIO_LIAPP-CTF{beta} oligomerization drives a presynaptic gain-of-function linked to neuronal hyperactivity C_LI

neuroscience↗

bioRxiv2025

Single-molecule analysis of synaptic protein complexes and vesicle recruitment

Single-molecule pull-down (SIM-Pull) combined with TIRF microscopy enables direct visualization of proteins and multi-protein complexes. Here, we present an extended SIM-Pull protocol for analyzing protein interactions at the active zone and their ability to recruit isolated synaptic vesicles (SV). SV recruitment mediated by STX1A-SNARE or RIM1-Rab3a interactions, respectively; can be directly visualized and quantified. This technique opens new avenues to examine the subcellular vesicle-associated protein-protein interactions at a molecular level in a near-native cellular context. HighlightsO_LIExtended SIM-Pull protocol combining biochemical isolation with TIRF microscopy to study synaptic protein complexes at near-native environment C_LIO_LIEnables direct quantification of synaptic vesicle recruitment at the surface (active zone) via protein-mediated vesicle tethering C_LIO_LIAdaptable platform for probing molecular interactions of protein complexes within different neuronal, cellular or subcellular compartments C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/671146v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1c42949org.highwire.dtl.DTLVardef@1727b9aorg.highwire.dtl.DTLVardef@110ec6aorg.highwire.dtl.DTLVardef@114ec7_HPS_FORMAT_FIGEXP M_FIG C_FIG Institutional permissionsAnimals were handled and maintained according to the guidelines laid down by the Animal Welfare Body (AWB) (Instantie voor Dierenwelzijn IvD) in line with the animal experimentation policy within Radboud University and RadboudUMC; under the license/protocol numbers 2021-0040-001/002 to Dr. Anne-Sophie Hafner.

molecular biology↗

bioRxiv2025

Amyloidogenic proteolysis of APP regulates glutamatergic presynaptic function

Disease causing mutations of Alzheimers disease (AD) point to dysregulations of APP proteolysis. During asymptomatic and early stages of AD, brain recordings revealed hyperexcitation reverting into over-inhibition as dementia progresses. Here, we show that endogenous APP and its proteolytic product APP-CTF{beta}, the precursors of A{beta}, accumulate preferentially at excitatory synapses. Using pharmacological treatments to modulate physiological concentrations of APP-CTF{beta} and A{beta}, we identify APP-CTF{beta} as a key regulator of glutamatergic synaptic transmission. Accumulation of APP-CTF{beta} increases the release probability of synaptic vesicles. Strikingly, monomeric A{beta} counteracts this APP-CTF{beta}-driven hyperexcitability. This suggests that therapeutic strategies clearing monomeric A{beta} could be detrimental during the early hyperexcitability phase of AD.

neuroscience↗