Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/066779

Bright photoactivatable fluorophores for single-molecule imaging

Luke D Lavis·Jonathan B Grimm·Brian P English·Anand K Muthusamy·Brian P Mehl·Peng Dong·Timothy A Brown·Zhe Liu·Timothée Lionnet
View original source ↗Export metadata ↓

Abstract

Small molecule fluorophores are important tools for advanced imaging experiments. The development of self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in live-cell microscopy. We recently described a general method for improving the brightness and photostability of small, cell-permeable fluorophores, resulting in the novel azetidine-containing \"Janelia Fluor\" (JF) dyes. Here, we refine and extend the utility of the JF dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. These compounds retain the superior brightness of the JF dyes once activated, but their facile photoactivation also enables improved single-particle tracking and localization microscopy experiments.

Source connections

Explore related subjects

Cell BiologyPreprint

Keep this discovery

BibTeXRIS

Luke D Lavis, Jonathan B Grimm, Brian P English, Anand K Muthusamy, Brian P Mehl, Peng Dong, Timothy A Brown, Zhe Liu, Timothée Lionnet. 2016-07-29. Bright photoactivatable fluorophores for single-molecule imaging. https://doi.org/10.1101/066779

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2016

Microtubule stabilization drives centrosome migration to initiate primary ciliogenesis

Primary cilia are sensory organelles located at the cell surface. Their assembly is primed by centrosome migration to the apical surface. Yet surprisingly little is known about this initiating step. To gain insight into the mechanisms driving centrosome migration, we exploited the reproducibility of cell architecture on adhesive micropatterns to investigate the cytoskeletal remodeling supporting it. Microtubule network densification and bundling, with the transient formation of an array of cold-stable microtubules, and actin cytoskeleton asymmetric contraction participated in concert to destabilize basal centrosome position and drive apical centrosome migration. The distal appendage protein Cep164 appeared to be a key actor involved in the cytoskeleton remodeling and centrosome migration, whereas IFT88's role seemed to be restricted to axoneme elongation. Together our data elucidate the hitherto unexplored mechanism of centrosome migration and show that it is driven by the increase and clustering of mechanical forces to push the centrosome toward the cell apical pole.

Cell Biology↗
bioRxiv2016

The duration of mitosis and daughter cell size are modulated by nutrients in budding yeast

The size of nearly all cells is modulated by nutrients. Thus, cells growing in poor nutrients can be nearly half the size of cells in rich nutrients. In budding yeast, cell size is thought to be controlled almost entirely by a mechanism that delays cell cycle entry until sufficient growth has occurred in G1 phase. Here, we show that most growth of a new daughter cell occurs in mitosis. When the rate of growth is slowed by poor nutrients, the duration of mitosis is increased, which suggests that cells compensate for slow growth in mitosis by increasing the duration of growth. The amount of growth required to complete mitosis is reduced in poor nutrients, leading to a large reduction in cell size. Together, these observations suggest that mechanisms that control the extent of growth in mitosis play a major role in cell size control in budding yeast.

Cell Biology↗
bioRxiv2016

Size-Dependent Accumulation of the Mitotic Activator Cdc25 as a Mechanism of Size Control in Fission Yeast

Proper cell size is essential for cellular function (Hall et al., 2004). Nonetheless, despite more than 100 years of work on the subject, the mechanisms that maintain cell size homeostasis are largely mysterious (Marshall et al., 2012). Cells in growing populations maintain cell size within a narrow range by coordinating growth and division. Bacterial and eukaryotic cells both demonstrate homeostatic size control, which maintains population-level variation in cell size within a certain range, and returns the population average to that range if it is perturbed (Marshall et al., 2012; Turner et al., 2012; Amodeo and Skotheim, 2015). Recent work has proposed two different strategies for size control: budding yeast has been proposed to use an inhibitor-dilution strategy to regulate size at the G1/S transition (Schmoller et al., 2015), while bacteria appear to use an adder strategy, in which a fixed amount of growth each generation causes cell size to converge on a stable average, a mechanism also suggested for budding yeast (Campos et al., 2014; Jun and Taheri-Araghi, 2015; Taheri-Araghi et al., 2015; Tanouchi et al., 2015; Soifer et al., 2016). Here we present evidence that cell size in the fission yeast Schizosaccharomyces pombe is regulated by a third strategy: the size dependent expression of the mitotic activator Cdc25. The cdc25 transcript levels are regulated such that smaller cells express less Cdc25 and larger cells express more Cdc25, creating an increasing concentration of Cdc25 as cell grow and providing a mechanism for cell to trigger cell division when they reach a threshold concentration of Cdc25. Since regulation of mitotic entry by Cdc25 is well conserved, this mechanism may provide a wide spread solution to the problem of size control in eukaryotes.

Cell Biology↗