High-affinity Tuning of Single Fluorescent Protein-type Indicators by Flexible Linker Length Optimization in Topology Mutant

Overview

Journal Commun Biol

Specialty Biology

Date 2024 Jun 8

PMID 38851844

Authors

Yusuke Hara

Aya Ichiraku

Tomoki Matsuda

Ayuko Sakane

Takuya Sasaki

Takeharu Nagai

Kazuki Horikawa

Affiliations

Soon will be listed here.

Abstract

Genetically encoded Ca indicators (GECIs) are versatile for live imaging of cellular activities. Besides the brightness and dynamic range of signal change of GECIs, Ca affinity is another critical parameter for successful Ca imaging, as the concentration range of Ca dynamics differs from low nanomolar to sub-millimolar depending on the celltype and organism. However, ultrahigh-affinity GECIs, particularly the single fluorescent protein (1FP)-type, are lacking. Here, we report a simple strategy that increases Ca affinity through the linker length optimization in topology mutants of existing 1FP-type GECIs. The resulting ultrahigh-affinity GECIs, CaMPARI-nano, BGECO-nano, and RCaMP-nano (K = 17-25 nM), enable unique biological applications, including the detection of low nanomolar Ca dynamics, highlighting active signaling cells, and multi-functional imaging with other second messengers. The linker length optimization in topology mutants could be applied to other 1FP-type indicators of glutamate and potassium, rendering it a widely applicable technique for modulating indicator affinity.

Citing Articles

A Sensitive Soma-localized Red Fluorescent Calcium Indicator for Multi-Modality Imaging of Neuronal Populations In Vivo.

Zhou S, Zhu Q, Eom M, Fang S, Subach O, Ran C bioRxiv. 2025; .

PMID: 39975286 PMC: 11838422. DOI: 10.1101/2025.01.31.635851.

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