Biology subjects

Preciado Lopez, M.

Publications and source records attributed to Preciado Lopez, M..

3 recordsLinked to original sources

bioRxiv2026

Triplet tumbling microscopy enables in situ quantification of protein complex assembly and dynamics

Protein-protein interactions (PPIs) mediate diverse cellular processes, but PPIs are typically characterized using reconstituted in vitro biochemical and biophysical approaches. Current approaches for PPI detection in living cells are limited in the scope of interactions they can capture and often require prior knowledge of the interacting partners. To close this gap, we developed triplet tumbling microscopy (TTM), which reveals the interactions of a tagged protein of interest in cells in real time. TTM reports protein complex size from rotational diffusion ("tumbling") by leveraging infrared-triggerable emission from triplet states to track tumbling over nanoseconds to hundreds of microseconds. These long-lived triplets overcome the size limitations of existing rotational diffusion-based approaches, enabling TTM to measure species from small protein complexes to organelle-scale beads. In living cells, we apply TTM to detect PPIs, quantify fraction bound, and distinguish protein complexes by size. We measure diverse types of interactions, including rapamycin-induced dimerization, p53 homo-oligomerization, and binding of the E3-ligase E6AP to the human papilloma virus 16 E6 protein. The required hardware is compatible with most fluorescent microscopes, making TTM a versatile way to extract molecular insights from the complex context of living cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/723557v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@c77bc1org.highwire.dtl.DTLVardef@8de44forg.highwire.dtl.DTLVardef@18803f2org.highwire.dtl.DTLVardef@bb3afe_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Branched multimeric peptides as affinity reagents for detection of α-Klotho protein

-Klotho is a protein associated with aging that is expressed in the kidney, parathyroid gland, and choroid plexus. As a transmembrane protein, it acts as an essential co-receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. -Klotho has an extracellular domain that can be cleaved, released and circulated in the blood stream as a soluble form. Decreased levels of -Klotho are an indication of chronic kidney disease and other age-associated diseases. Detecting or labeling transmembrane and soluble -Klotho is a longstanding challenge that has impeded the in-depth understanding of its role. Here we describe branched multimeric peptides that recognize -Klotho with high affinity and selectivity in the biological milieu. The branched peptides are prepared in a single-shot synthesis by parallel automated fast-flow synthesis in under one hour. The branched -Klotho-binding peptides show improvement in affinity relative to the monomeric versions and can be used to label Klotho for live imaging in kidney cells. Our results demonstrate the potential of automated flow technology to deliver peptide-based reagents with complex architecture and improved affinity for the selective binding of target proteins in physiological settings.

molecular biology↗

bioRxiv2019

Mechanisms of motor-independent membrane remodeling driven by dynamic microtubules

Microtubule-dependent organization of membranous organelles, such as the endoplasmic reticulum, occurs through motor-based pulling and by coupling microtubule dynamics to membrane remodeling. How highly transient protein-protein interactions occurring at growing microtubule tips can induce load-bearing processive motion is currently unclear. Here, we reconstituted membrane tubulation in a minimal system with giant unilamellar vesicles, dynamic microtubules, End-Binding (EB) proteins and a membrane-targeted protein that interacts with EBs and microtubules. We showed that these components are sufficient to drive membrane remodeling by three mechanisms: membrane tubulation by growing microtubule ends, motor-independent membrane sliding along microtubule shafts and pulling by shrinking microtubules. Experiments and modeling demonstrated that the first two mechanisms can be explained by adhesion-driven biased membrane spreading on microtubules. Force spectroscopy revealed that attachments to growing and shrinking microtubule ends can sustain forces of [~]0.5 and [~]5 pN, respectively. Rapidly exchanging molecules that connect membranes to dynamic microtubules can thus bear sufficient load to induce membrane deformation and motility.

cell biology↗