Arakawa, M. | bioRxiv Science

bioRxiv2024

A Novel Method to Visualize Active Small GTPases Unveils Distinct Sites of Sar1 Activation During Collagen Secretion.

Small GTPases are essential in various cellular signaling pathways, and detecting their activation within living cells is crucial for understanding cellular processes. Fluorescence resonance energy transfer is widely used to study the interaction between activated small GTPases and their effectors, but it is limited to those with well-defined effectors, excluding Sar1. Here, we present a novel method, SAIYAN (Small GTPase ActIvitY ANalyzing), for detecting the activation of endogenous small GTPases via fluorescent signals utilizing a split mNeonGreen system. We demonstrated Sar1 activation at the endoplasmic reticulum (ER) exit site and successfully detected its activation state in various cellular conditions. Utilizing the SAIYAN system in collagen-secreting cells, we discovered activated Sar1 localized both at ER exit sites and ER-Golgi intermediate compartment (ERGIC) regions. Additionally, impaired collagen secretion led to the confinement of activated Sar1 at the ER exit sites, underscoring the significance of Sar1 activation through the ERGIC in collagen secretion.

cell biology↗

bioRxiv2021

Deceleration of cell cycle underpins a switch from proliferative- to terminal division in plant stomatal lineage

Differentiation of specialized cell types from self-renewing progenitors requires precise cell cycle control. Plant stomata are generated through asymmetric divisions of a stem-cell-like precursor meristemoid followed by the single symmetric division that creates an adjustable pore surrounded by paired guard cells. The stomatal-lineage-specific transcription factor MUTE terminates the asymmetric divisions and triggers differentiation. However, the role of cell cycle machinery in this transition remains unknown. Through time-lapse imaging, we discover that the symmetric division is slower than the asymmetric division. We identify a plant-specific cyclin-dependent kinase inhibitor, SIAMESE-RELATED4 (SMR4), as a molecular brake that decelerates cell cycle during this transition. SMR4 is directly induced by MUTE and transiently accumulates in differentiating meristemoids. SMR4 physically and functionally associates with CYCD3;1 and extends G1-phase of asymmetric divisions. By contrast, SMR4 fails to interact with CYCD5;1, a MUTE-induced G1 cyclin, and permits the symmetric division. Our work unravels a molecular framework of the proliferation-to-differentiation switch within the stomatal lineage and suggests that a timely proliferative cell cycle is critical for the stomatal fate specification.

plant biology↗