Yu-Chun Lin

The CP110-interacting proteins Talpid3 and Cep290 play overlapping and distinct roles in cilia assembly

Talpid3 and Cep290 promote proper ciliary vesicle formation by regulating centriolar satellite accretion and Rab8a localization.

Rapidly reversible manipulation of molecular activity with dual chemical dimerizers.

The rapid, inducible, reversible modulation of molecular activities by dual CID systems. Rapamycin (Rapa) treatment induces relocation of FRB-POI to the GAIs-FKBP-C2(LACT)-labeled plasma membrane and activates POI-dependent signaling event. A subsequent GA3-AM treatment induces second relocation of a whole GAIs-FKBP-C2(LACT)/rapamycin/FRB-POI complex from the plasma membrane to the Tom20-GID1-labeled mitochondria and leads to termination of POI-dependent signaling.

An Intelligent Nano-Antenna: Primary cilium harnesses TRP channels to decode polymodal stimuli

The primary cilium is a solitary hair-like organelle on the cell surface that serves as an antenna sensing ever-changing environmental conditions. In this review, we will first recapitulate the molecular basis of the polymodal sensory function of the primary cilia, specifically focusing on transient receptor potential (TRP) channels that accumulate inside the organelle and conduct calcium ions (Ca(2+)). Each subfamily member, namely TRPP2 TRPP3, TRPC1 and TRPV4, is gated by multiple environmental factors, including chemical (receptor ligands, intracellular second messengers such as Ca(2+)), mechanical (fluid shear stress, hypo-osmotic swelling), or physical (temperature, voltage) stimuli. Both activity and heterodimer compositions of the TRP channels may be dynamically regulated for precise tuning to the varying dynamic ranges of the individual input stimuli. We will thus discuss the potential regulation of TRP channels by local second messengers. Despite its reported importance in embryonic patterning and tissue morphogenesis, the precise functional significance of the downstream Ca(2+) signals of the TRP channels remains unknown. We will close our review by featuring recent technological advances in visualizing and analyzing signal transduction inside the primary cilia, together with current perspectives illuminating the functional significance of intraciliary Ca(2+) signals.

The small GTPase HRas shapes local PI3K signals through positive feedback and regulates persistent membrane extension in migrating fibroblasts

An inducible activation approach is used to interrogate the positive feedback circuit underlying self-amplification of PI3K signals in fibroblasts. The results show that local positive feedback between PI3K and HRas drives asymmetric membrane extension and cell migration.

Angiogenesis-targeting microbubbles combined with ultrasound-mediated gene therapy in brain tumors

&NA; The major challenges in gene therapy for brain cancer are poor transgene expression due to the blood‐brain barrier (BBB) and neurologic damage caused by conventional intracerebral injection. Non‐viral gene delivery using ultrasound‐targeted microbubbles (MBs) oscillation via the systematic transvascular route is attractive, but there is currently no high‐yielding and targeted gene expression method. In this study, we developed a non‐viral and angiogenesis‐targeting gene delivery approach for efficient brain tumor gene therapy without brain damage. We developed a VEGFR2‐targeted and cationic microbubbles (VCMBs) gene vector for use with transcranial focused ultrasound (FUS) exposure to allow transient gene delivery. The system was tested in a brain tumor model using the firefly luciferase gene and herpes simplex virus type 1 thymidine kinase/ganciclovir (pHSV‐TK/GCV) with VCMBs under FUS exposure for transgene expression and anti‐tumor effect. In vitro data showed that VCMBs have a high DNA‐loading efficiency and high affinity for cancer cells. In vivo data confirmed that this technique enhanced gene delivery into tumor tissues without affecting normal brain tissues. The VCMBs group resulted in higher luciferase expression (3.8 fold) relative to the CMBs group (1.9 fold), and the direct injection group. The tumor volume on day 25 was significantly smaller in rats treated with the pHSV‐TK/GCV system using VCMBs under FUS (9.7 ± 5.2 mm3) than in the direct injection group (40.1 ± 4.3 mm3). We demonstrated the successful use of DNA‐loaded VCMBs and FUS for non‐viral, non‐invasive and targeted gene delivery to brain tumors. Graphical abstract Figure. No caption available.

Visualizing Molecular Diffusion through Passive Permeability Barriers in Cells: Conventional and Novel Approaches

Diffusion barriers are universal solutions for cells to achieve distinct organizations, compositions, and activities within a limited space. The influence of diffusion barriers on the spatiotemporal dynamics of signaling molecules often determines cellular physiology and functions. Over the years, the passive permeability barriers in various subcellular locales have been characterized using elaborate analytical techniques. In this review, we will summarize the current state of knowledge on the various passive permeability barriers present in mammalian cells. We will conclude with a description of several conventional techniques and one new approach based on chemically inducible diffusion trap (CIDT) for probing permeable barriers.

Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling

Tubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells and are suggested to be involved in a wide range of cellular activities. However, the complexity and dynamic distribution of tubulin PTMs within cells have hindered the understanding of their physiological roles in specific subcellular compartments. Here, we develop a method to rapidly deplete tubulin glutamylation inside the primary cilia, a microtubule-based sensory organelle protruding on the cell surface, by targeting an engineered deglutamylase to the cilia in minutes. This rapid deglutamylation quickly leads to altered ciliary functions such as kinesin-2-mediated anterograde intraflagellar transport and Hedgehog signaling, along with no apparent crosstalk to other PTMs such as acetylation and detyrosination. Our study offers a feasible approach to spatiotemporally manipulate tubulin PTMs in living cells. Future expansion of the repertoire of actuators that regulate PTMs may facilitate a comprehensive understanding of how diverse tubulin PTMs encode ciliary as well as cellular functions.Tubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells, therefore assessing the physiological roles in specific subcellular compartments has been challenging. Here the authors develop a method to rapidly deplete tubulin glutamylation inside the primary cilia by targeting an engineered deglutamylase to the axoneme.

Ultrasound-chemical hybrid system for manipulating cellular activities

Focused ultrasound (FUS) with microbubbles (MBs) can enhance delivery of impermeable materials into cells for eliminating abnormal cells or enhancing gene transfection. Very few of the existing systems can precisely control cellular physiology. Chemically-inducible dimerization (CID) system has already gained widespread popularity as a tool in cell biology and has been used to address the diverse, yet fundamental biological questions. Although remotely triggering CID system by external stimuli such as light has been developed, it is still challenging to deliver light to the tissues deeper than just 2 mm without any invasive surgical procedures. This study proposed a new ultrasound-chemical hybrid system to trigger the CID system by FUS with MBs. With FUS, membrane impermeable chemical dimerizers can be introduced into cells to spatiotemporally control cellular physiology within timescale of minutes.

Intracellular production of hydrogels and synthetic RNA granules by multivalent enhancers

Non-membrane bound, hydrogel-like entities, such as RNA granules, nucleate essential cellular functions through their unique physico-chemical properties. However, these intracellular hydrogels have not been as extensively studied as their extracellular counterparts, primarily due to technical challenges in probing these materials in situ. Here, by taking advantage of a chemically inducible dimerization paradigm, we developed iPOLYMER, a strategy for rapid induction of protein-based hydrogels inside living cells. A series of biochemical and biophysical characterizations, in conjunction with computational modeling, revealed that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that behaves as a size-dependent molecular sieve. We studied several properties of the gel and functionalized it with RNA binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. Therefore, we here demonstrate that iPOLYMER presents a unique and powerful approach to synthetically reconstitute hydrogel-like structures including RNA granules in intact cells.

Manipulating cellular activities using an ultrasound-chemical hybrid tool

We developed an ultrasound-chemical hybrid tool to precisely manipulate cellular activities. A focused ultrasound coupled with gas-filled microbubbles was used to rapidly trigger the influx of membrane-impermeable chemical dimerizers into living cells to regulate protein dimerization and location without inducing noticeable toxicity. With this system, we demonstrated the successful modulation of phospholipid metabolism triggered by a short pulse of ultrasound exposure. Our technique offers a powerful and versatile tool for using ultrasound to spatiotemporally manipulate the cellular physiology in living cells.