Biology subjects

Kallem, T.

Publications and source records attributed to Kallem, T..

4 recordsLinked to original sources

bioRxiv2026

An internal PDZ-binding motif in Densin-180 promotes activity-dependent SHANK scaffold remodelling

SHANK proteins form core postsynaptic density (PSD) scaffolds that organise synaptic signalling complexes through multiple protein-protein interaction domains, including PDZ domains that typically recognise C-terminal peptide motifs. Here, we identify an internal PDZ recognition mechanism that links the synaptic protein Densin-180 to SHANK and promotes SHANK scaffold assembly. We map SHANK binding to an internal PDZ-binding motif in Densin-180 (residues 843-863) and show by NMR spectroscopy and fluorescence polarisation that this motif binds SHANK1-3 PDZ domains with high affinity and specificity, competing with canonical C-terminal ligands. The crystal structure of the Densin-180-SHANK complex reveals that Phe858 inserts into the hydrophobic pocket of the PDZ domain despite the absence of a terminal carboxylate. In neurons, this interaction mediates Densin-180 recruitment to dendritic spines and drives activity-dependent reorganisation of postsynaptic SHANK3 assemblies, whereas mutation of Phe858 disrupts both processes. Disrupting the Densin-180-SHANK interaction using a Densin-180-derived peptide impairs activity-dependent structural plasticity of spines, accompanied by defects in PSD organisation and actin cytoskeleton remodelling. These findings define an internal mode of PDZ recognition and reveal how Densin-180 couples neuronal activity to the remodelling of postsynaptic SHANK scaffolds.

neuroscience↗

bioRxiv2026

The KN domain of KANK proteins contains separable talin-binding and intramolecular interaction modules

KANK proteins link integrin adhesions to the cortical microtubule stabilising complex (CMSC) through interactions with the adhesion adaptor talin. However, how KANK proteins are regulated remains unclear. Here we show that the KN domain of KANK proteins contains separable regions that mediate talin binding and a conserved intramolecular interaction. Using fluorescence polarisation, NMR spectroscopy and structural analysis, we map an interaction between the N-terminal KN domain and the C-terminal ankyrin repeat domain and identify residues 60-68 of the KN domain as required for this intramolecular interaction. In contrast, the canonical LD motif within residues 30-60 mediates binding to talin. Deletion of residues 60-68 disrupts the intramolecular interaction while preserving talin binding, demonstrating that the KN domain contains distinct modules for talin engagement and intramolecular regulation. This regulatory architecture is conserved across the KANK family, although sequence variation modulates the strength of the intramolecular interaction. Together, these findings identify a modular organisation within the KANK KN domain that separates talin recognition from intramolecular regulation and is consistent with an autoinhibitory mechanism. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/731086v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@e2dd22org.highwire.dtl.DTLVardef@240996org.highwire.dtl.DTLVardef@1f1926borg.highwire.dtl.DTLVardef@c3e523_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO The KN domain of KANK proteins contains separable talin-binding and intramolecular interaction modules.The LD motif (blue) mediates binding to the talin R7 domain, whereas residues 60-68 (yellow) are required for interaction with the C-terminal ankyrin repeat domain. An AlphaFold model is shown as a structural interpretation of the intramolecular KN-ankyrin repeat interaction identified in this study. C_FIG

biochemistry↗

bioRxiv2026

Talin controls the spatial distribution of vinculin tension in focal adhesions

Cells transmit force between the extracellular matrix and the actin cytoskeleton through integrin adhesion complexes centred on talin and vinculin. Vinculin binds talin through -helical vinculin-binding sites (VBS) that are exposed when talin rod domains unfold under force. Dissecting the significance of this interaction has relied heavily on the A50I mutation in vinculin, which has been widely used as a talin-binding-null mutant. Here we show that although the A50I mutation abolishes binding to the -catenin VBS, it retains nanomolar-affinity binding to multiple talin VBS. We therefore designed an improved mutant, I12K/A50I, that eliminates this residual talin binding. Biochemical assays and single-molecule stretching experiments demonstrate that I12K/A50I VD1 fails to bind talin even when VBS are exposed by force. Using vinculin tension and conformation sensors, we show that talin binding is required for efficient recruitment of vinculin to focal adhesions and for establishing spatial gradients of vinculin tension. However, vinculin can still experience mechanical load in the absence of talin binding. These results demonstrate that A50I is not a talin-binding-null and reveal that, while talin is not required for vinculin loading, it is essential for organising the spatial distribution of mechanical load within adhesion complexes.

cell biology↗

bioRxiv2024

Glycogen phase separation drives macromolecular rearrangement and asymmetric division in E. coli

Bacteria often experience nutrient limitation. While the exponential and stationary growth phases have been characterized in the model bacterium Escherichia coli, little is known about what transpires inside individual cells during the transition between these two phases. Through quantitative cell imaging, we found that the positions of nucleoids and cell division sites become increasingly asymmetric during transition phase. These asymmetries were coupled with asymmetric reorganization of protein, ribosome, and RNA probes in the cytoplasm. Results from live-cell imaging experiments, complemented with genetic and 13C whole-cell nuclear magnetic resonance spectroscopy studies, show that preferential accumulation of the storage polymer glycogen at the old cell pole leads to the observed rearrangements and asymmetric divisions. Live-cell atomic force microscopy analysis, combined with in vitro biochemical experiments, suggests that these phenotypes are due to the propensity of glycogen to phase separate into soft condensates in the crowded cytoplasm. Glycogen-associated differences in cell sizes between strains and future daughter cells suggest that glycogen phase separation allows cells to store large glucose reserves without counting them as cytoplasmic space.

microbiology↗