Biology subjects

Chen, Y.-J.

Publications and source records attributed to Chen, Y.-J..

5 recordsLinked to original sources

bioRxiv2018

Glucose intake hampers PKA-regulated HSP90 chaperone activity

Aging is an intricate phenomenon associated with the gradual loss of physiological functions, and both nutrient sensing and proteostasis control lifespan. Although multiple approaches have facilitated the identification of candidate genes that govern longevity, the molecular mechanisms that link aging pathways are still elusive. Here, we conducted a quantitative mass spectrometry screen and identified all phosphorylation/dephosphorylation sites on yeast proteins that significantly responded to calorie restriction, a well-established approach to extend lifespan. Functional screening of 135 potential regulators uncovered that Ids2 is activated by PP2C under CR and inactivated by PKA under glucose intake. ids2{Delta} or ids2 phosphomimetic cells displayed heat sensitivity and lifespan shortening. Ids2 serves as a co-chaperone to form a complex with Hsc82 or the redundant Hsp82, and phosphorylation of Ids2 impedes its association with chaperone HSP90. Thus, PP2C and PKA orchestrate glucose sensing and protein folding to enable cells to maintain protein quality for sustained longevity.

cell biology↗

bioRxiv2017

EB1 binding provides a diffusion trap mechanism regulating STIM1 localization and Ca2+ signaling

The endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER-plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) following ER Ca2+ depletion. STIM1 also directly interacts with end binding protein 1 (EB1) at microtubule (MT) plus-ends and resembles comet-like structures during time-lapse imaging. Nevertheless, the role of STIM1-EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with pharmacological perturbation and a reconstitution approach, we revealed that EB1 binding constitutes a diffusion trap mechanism restricting STIM1 targeting to ER-PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. EB1 binding delayed the translocation of STIM1 oligomers to ER-PM junctions and recaptured STIM1 to prevent excess SOCE and ER Ca2+ overload. Thus, the counterbalance of EB1 binding and PM targeting of STIM1 shapes the kinetics and amplitude of local SOCE in regions with growing MTs, and contributes to precise spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.\n\nSummarySTIM1 activates store-operated Ca2+ entry (SOCE) by translocating to endoplasmic reticulum-plasma membrane junctions. Chang et al. revealed that STIM1 localization and SOCE are regulated by a diffusion trap mechanism mediated by STIM1 binding to EB1 at growing microtubule ends.

cell biology↗

bioRxiv2017

Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in

CRISPR-based gene knock-in at endogenous sites is desirable in multiple fields such as quantitative studies of signal transduction pathways and gene regulation, synthetic biology, and disease modeling. Contrasting the knock-out procedure, a key step of CRISPR knock-in procedure relies on the homology-directed repairing (HDR) process that requires a donor construct as repair template. Therefore, it is desirable to generate a series of donor DNA constructs efficiently and cost-effectively. In this study, we developed a general Gibson assembly procedure that combines strengths of a Modular Overlap-Directed Assembly with Linkers (MODAL) strategy and a restriction enzyme based hierarchical framework. This procedure also allows fusing sgRNAs to the constructs for enhanced homology-directed repairing efficiency. Experimental tests on multiple constructs achieved from 3-8 folds of increase in assembly efficiency to high yield of constructs that failed to make with conventional Gibson assembly. The modularized procedure is simple, fast and cost-effective while making multiple constructs, and a computer package is provided for customized design.

molecular biology↗

bioRxiv2017

Cells Interpret Temporal Information From TGF-β Through A Nested Relay Mechanism

The detection and transmission of the temporal quality of intracellular and extracellular signals is an essential cellular mechanism. It remains largely unexplored how cells interpret the duration information of a stimulus. In this paper, through an integrated quantitative and computational approach we demonstrate that crosstalk among multiple TGF-{beta} activated pathways forms a relay from SMAD to GLI1 that initializes and maintains SNAILl expression, respectively. This transaction is smoothed and accelerated by another temporal switch from elevated cytosolic GSK3 enzymatic activity to reduced nuclear GSK3 enzymatic activity. The intertwined network places SNAIL1 as a key integrator of information from TGF-{beta} signaling subsequently distributed through upstream divergent pathways; essentially cells generate a transient or sustained expression of SNAIL1 depending on TGF-{beta} duration. Other signaling pathways may use similar network structure to encode duration information.

systems biology↗

bioRxiv2017

Scaling up DNA data storage and randomaccess retrieval

Current storage technologies can no longer keep pace with exponentially growing amounts of data. 1 Synthetic DNA offers an attractive alternative due to its potential information density of ~ 1018 B/mm3, 107 times denser than magnetic tape, and potential durability of thousands of years.2 Recent advances in DNA data storage have highlighted technical challenges, in particular, coding and random access, but have stored only modest amounts of data in synthetic DNA. 3,4,5 This paper demonstrates an end-to-end approach toward the viability of DNA data storage with large-scale random access. We encoded and stored 35 distinct files, totaling 200MB of data, in more than 13 million DNA oligonucleotides (about 2 billion nucleotides in total) and fully recovered the data with no bit errors, representing an advance of almost an order of magnitude compared to prior work. 6 Our data curation focused on technologically advanced data types and historical relevance, including the Universal Declaration of Human Rights in over 100 languages,7 a high-definition music video of the band OK Go,8 and a CropTrust database of the seeds stored in the Svalbard Global Seed Vault.9 We developed a random access methodology based on selective amplification, for which we designed and validated a large library of primers, and successfully retrieved arbitrarily chosen items from a subset of our pool containing 10.3 million DNA sequences. Moreover, we developed a novel coding scheme that dramatically reduces the physical redundancy (sequencing read coverage) required for error-free decoding to a median of 5x, while maintaining levels of logical redundancy comparable to the best prior codes. We further stress-tested our coding approach by successfully decoding a file using the more error-prone nanopore-based sequencing. We provide a detailed analysis of errors in the process of writing, storing, and reading data from synthetic DNA at a large scale, which helps characterize DNA as a storage medium and justify our coding approach. Thus, we have demonstrated a significant improvement in data volume, random access, and encoding/decoding schemes that contribute to a whole-system vision for DNA data storage.

bioengineering↗