Semiconductor small molecule IHIC/ITIC applied to photothermal therapy and photoacoustic imaging of tumors.

Organic semiconductor small molecules IHIC and ITIC have been developed as solar cell materials, and because of their strong near-infrared absorption capabilities, they are promising for cancer phototherapy. This article reports the application of semiconductor small molecule IHIC/ITIC liposomes in photothermal therapy and photoacoustic imaging of tumors firstly. Experiments show that the liposome-loaded IHIC/ITIC material has good biocompatibility and can be effectively enriched in tumor sites. After being irradiated with laser, it can emit strong photoacoustic signals, and has achieved good results in the photothermal treatment of breast cancer mice. We believe that organic semiconductor small molecule IHIC/ITIC will become a promising photothermal agent with wonderful development possibilities.

Rab10-Positive Tubular Structures Represent a Novel Endocytic Pathway That Diverges From Canonical Macropinocytosis in RAW264 Macrophages.

Using the optogenetic photo-manipulation of photoactivatable (PA)-Rac1, remarkable cell surface ruffling and the formation of a macropinocytic cup (premacropinosome) could be induced in the region of RAW264 macrophages irradiated with blue light due to the activation of PA-Rac1. However, the completion of macropinosome formation did not occur until Rac1 was deactivated by the removal of the light stimulus. Following PA-Rac1 deactivation, some premacropinosomes closed into intracellular macropinosomes, whereas many others transformed into long Rab10-positive tubules without forming typical macropinosomes. These Rab10-positive tubules moved centripetally towards the perinuclear Golgi region along microtubules. Surprisingly, these Rab10-positive tubules did not contain any endosome/lysosome compartment markers, such as Rab5, Rab7, or LAMP1, suggesting that the Rab10-positive tubules were not part of the degradation pathway for lysosomes. These Rab10-positive tubules were distinct from recycling endosomal compartments, which are labeled with Rab4, Rab11, or SNX1. These findings suggested that these Rab10-positive tubules may be a part of non-degradative endocytic pathway that has never been known. The formation of Rab10-positive tubules from premacropinosomes was also observed in control and phorbol myristate acetate (PMA)-stimulated macrophages, although their frequencies were low. Interestingly, the formation of Rab10-positive premacropinosomes and tubules was not inhibited by phosphoinositide 3-kinase (PI3K) inhibitors, while the classical macropinosome formation requires PI3K activity. Thus, this study provides evidence to support the existence of Rab10-positive tubules as a novel endocytic pathway that diverges from canonical macropinocytosis.

Exosomes and exosomal microRNA in non-targeted radiation bystander and abscopal effects in the central nervous system.

Localized cranial radiotherapy is a dominant treatment for brain cancers. After being subjected to radiation, the central nervous system (CNS) exhibits targeted effects as well as non-targeted radiation bystander effects (RIBE) and abscopal effects (RIAE). Radiation-induced targeted effects in the CNS include autophagy and various changes in tumor cells due to radiation sensitivity, which can be regulated by microRNAs. Non-targeted radiation effects are mainly induced by gap junctional communication between cells, exosomes containing microRNAs can be transduced by intracellular endocytosis to regulate RIBE and RIAE. In this review, we discuss the involvement of microRNAs in radiation-induced targeted effects, as well as exosomes and/or exosomal microRNAs in non-targeted radiation effects in the CNS. As a target pathway, we also discuss the Akt pathway which is regulated by microRNAs, exosomes, and/or exosomal microRNAs in radiation-induced targeted effects and RIBE in CNS tumor cells. As the CNS-derived exosomes can cross the blood-brain-barrier (BBB) into the bloodstream and be isolated from peripheral blood, exosomes and exosomal microRNAs can emerge as promising minimally invasive biomarkers and therapeutic targets for radiation-induced targeted and non-targeted effects in the CNS.

Enhanced Internalization of Nanoparticles Following Ionizing Radiation Leads to Mitotic Catastrophe in MG-63 Human Osteosarcoma Cells.

This study aims to investigate whether ionizing radiation combined with doxorubicin-conjugated iron oxide nanoparticles (NP-DOX) improves the internalization and cytotoxic effects of the nano-carrier-mediated drug delivery in MG-63 human osteosarcoma cells. NP-DOX was designed and synthesized using the co-precipitation method. Highly stable and crystalline nanoparticles conjugated with DOX were internalized in MG-63 cells through macropinocytosis and located in the perinuclear area. Higher nanoparticles internalization in MG-63 cells previously exposed to 1 Gy X-rays was correlated with an early accumulation of cells in G2/M, starting at 12 h after treatment. After 48 h, the application of the combined treatment led to higher cytotoxic effects compared to the individual treatment, with a reduction in the metabolic capacity and unrepaired DNA breaks, whilst a low percent of arrested cells, contributing to the commitment of mitotic catastrophe. NP-DOX showed hemocompatibility and no systemic cytotoxicity, nor histopathological alteration of the main organs.

Interplay between Cellular Uptake, Intracellular Localization and the Cell Death Mechanism in Triphenylamine-Mediated Photoinduced Cell Death.

Triphenylamines (TPAs) were previously shown to trigger cell death under prolonged one- or two-photon illumination. Their initial subcellular localization, before prolonged illumination, is exclusively cytoplasmic and they translocate to the nucleus upon photoactivation. However, depending on their structure, they display significant differences in terms of precise initial localization and subsequent photoinduced cell death mechanism. Here, we investigated the structural features of TPAs that influence cell death by studying a series of molecules differing by the number and chemical nature of vinyl branches. All compounds triggered cell death upon one-photon excitation, however to different extents, the nature of the electron acceptor group being determinant for the overall cell death efficiency. Photobleaching susceptibility was also an important parameter for discriminating efficient/inefficient compounds in two-photon experiments. Furthermore, the number of branches, but not their chemical nature, was crucial for determining the cellular uptake mechanism of TPAs and their intracellular fate. The uptake of all TPAs is an active endocytic process but two- and three-branch compounds are taken up via distinct endocytosis pathways, clathrin-dependent or -independent (predominantly caveolae-dependent), respectively. Two-branch TPAs preferentially target mitochondria and photoinduce both apoptosis and a proper necrotic process, whereas three-branch TPAs preferentially target late endosomes and photoinduce apoptosis only.

Core­shell type thermo­nanoparticles loaded with temozolomide combined with photothermal therapy in melanoma cells.

A novel core­shell type thermo­nanoparticle (CSTNP) co­loaded with temozolomide (TMZ) and the fluorescein new indocyanine green dye IR820 (termed IT­CSTNPs) was designed and combined with a near­infrared (NIR) laser to realize its photothermal conversion. The IT­CSTNPs were prepared using a two­step synthesis method and comprised a thermosensitive shell and a biodegradable core. IR820 and TMZ were entrapped in the shell and the core, respectively. Dynamic light scattering results demonstrated that the average hydrodynamic size of the IT­CSTNPs was 196.4±3.1 nm with a ζ potential of ­24.9±1.3 mV. The encapsulation efficiencies of TMZ and IR820 were 6.1 and 16.6%, respectively. Temperature increase curves under NIR laser irradiation indicated that the IT­CSTNPs exhibited the desired photothermal conversion efficiency. The in vitro drug release curves revealed a suitable release capability of IT­CSTNP under physiological conditions, whereas NIR laser irradiation accelerated the drug release. Inverted fluorescence microscopy and flow cytometry results revealed that the uptake of IT­CSTNPs by A375 melanoma cells occurred in a concentration­dependent manner. Confocal laser scanning microscopy results indicated that IT­CSTNPs entered tumour cells via endocytosis and were located in intercellular lysosomes. In summary, the present study explored the photothermal conversion capability, cellular uptake, and intracellular localization of IT­CSTNPs.

Secretory Vesicle Clustering in Fungal Filamentous Cells Does Not Require Directional Growth.

During symmetry breaking, the highly conserved Rho GTPase Cdc42 becomes stabilized at a defined site via an amplification process. However, little is known about how a new polarity site is established in an already asymmetric cell-a critical process in a changing environment. The human fungal pathogen Candida albicans switches from budding to filamentous growth in response to external cues, a transition controlled by Cdc42. Here, we have used optogenetic manipulation of cell polarity to reset growth in asymmetric filamentous C. albicans cells. We show that increasing the level of active Cdc42 on the plasma membrane results in disruption of the exocyst subunit Sec3 localization and a striking de novo clustering of secretory vesicles. This new cluster of secretory vesicles is highly dynamic, moving by hops and jumps, until a new growth site is established. Our results reveal that secretory vesicle clustering can occur in the absence of directional growth.

Rab5C enhances resistance to ionizing radiation in rectal cancer.

Rectal cancer represents one third of the colorectal cancers that are diagnosed. Neoadjuvant chemoradiation is a well-established protocol for rectal cancer treatment reducing the risk of local recurrence. However, a pathologic complete response is only achieved in 10-40% of cases and the mechanisms associated with resistance are poorly understood. To identify potential targets for preventing therapy resistance, a proteomic analysis of biopsy specimens collected from stage II and III rectal adenocarcinoma patients before neoadjuvant treatment was performed and compared with residual tumor tissues removed by surgery after neoadjuvant therapy. Three proteins, Ku70, Ku80, and Rab5C, exhibited a significant increase in expression after chemoradiation. To better understand the role of these proteins in therapy resistance, a rectal adenocarcinoma cell line was irradiated to generate a radiotherapy-resistant lineage. These cells overexpressed the same three proteins identified in the tissue samples. Furthermore, radiotherapy resistance in this in vitro model was found to involve modulation of epidermal growth factor receptor (EGFR) internalization by Rab5C in response to irradiation, affecting expression of the DNA repair proteins, Ku70 and Ku80, and cell resistance. Taken together, these findings indicate that EGFR and Rab5C are potential targets for the sensitization of rectal cancer cells and they should be further investigated. KEY MESSAGES: • Rab5C orchestrates a mechanism of radioresistance in rectal adenocarcinoma cell. • Rab5C modulates EGFR internalization and its relocalization to the nucleus. • In the nucleus, EGFR can modulate the expression of the DNA repair proteins, Ku70 and Ku80. • Rab5C, Ku70, and Ku80 are overexpressed in tumor tissues that contain tumor cells that are resistant to chemoradiation treatment.

Minireview: Biophysical Mechanisms of Cell Membrane Sonopermeabilization. Knowns and Unknowns.

Microbubble-assisted ultrasound has emerged as a promising method for the delivery of low-molecular-weight chemotherapeutic molecules, nucleic acids, therapeutic peptides, and antibodies in vitro and in vivo. Its clinical applications are under investigation for local delivery drug in oncology and neurology. However, the biophysical mechanisms supporting the acoustically mediated membrane permeabilization are not fully established. This review describes the present state of the investigations concerning the acoustically mediated stimuli (i.e., mechanical, chemical, and thermal stimuli) as well as the molecular and cellular actors (i.e., membrane pores and endocytosis) involved in the reversible membrane permeabilization process. The different hypotheses, which were proposed to give a biophysical description of the membrane permeabilization, are critically discussed.

An acoustic strategy for gold nanoparticle loading in platelets as biomimetic multifunctional carriers.

In recent years, a wide variety of bioinspired colloidal particles with novel cell mimetic functions have been the subject of extensive research in materials science, chemistry, biology, physics, and engineering. However, most of the approaches are derived from natural cell membrane coatings, which are still too primitive compared with living cells. In this study, we have chosen gold nanoparticles (GNPs) to explore the bioactivity response of living platelets and nanoparticle loading efficiency under different ultrasonic intensity and frequency treatment conditions. The results show that GNPs with no surface modification could be easily loaded into intra-platelets by both incubation (30 min) and ultrasonic exposure (1 min) methods. The amount of GNP loading was (4.4 ± 0.9) × 10-3 and (5.8 ± 2.4) × 10-3 pg per platelet upon incubation and acoustic triggering (1 MHz, 0.25 W cm-2), respectively. Although the other US treatment intensities (0.75, 1.50 and 2.25 W cm-2) also promoted higher amounts of GNPs in the platelets, the higher US intensity might bring about partial damage of the platelet membrane. Compared with 1 MHz ultrasonic exposure, the change of the GNP loading amount was not significantly higher upon ultrasonic frequency treatment of 45, 80 or 100 kHz. Therefore, it has been found that an US intensity of 0.25 W cm-2 could facilitate the intra-platelet delivery efficacy of the GNPs without damaging the biological activity. Furthermore, two possible pathways of GNPs entering into platelets upon US treatment are presented: one is the endocytosis/open canalicular system (OCS), and the other is cell membrane permeability enhancement, which is proved by the SEM and TEM results. Finally, the GNP-loaded platelets have been demonstrated as useful probes for photoacoustic imaging (PAI) and dark-field microscopy (DFM)-based imaging, which might allow a wide range of potential applications in diagnostics and therapy of platelet-related diseases.

Noninvasive 40-Hz light flicker to recruit microglia and reduce amyloid beta load.

Microglia, the primary immune cells of the brain, play a key role in pathological and normal brain function. Growing efforts aim to reveal how these cells may be harnessed to treat both neurodegenerative diseases such as Alzheimer's and developmental disorders such as schizophrenia and autism. We recently showed that using noninvasive exposure to 40-Hz white-light (4,000 K) flicker to drive 40-Hz neural activity transforms microglia into an engulfing state and reduces amyloid beta, a peptide thought to initiate neurotoxic events in Alzheimer's disease (AD). This article describes how to construct an LED-based light-flicker apparatus, expose animals to 40-Hz flicker and control conditions, and perform downstream assays to study the effects of these stimuli. Light flicker is simple, faster to implement, and noninvasive, as compared with driving 40-Hz activity using optogenetics; however, it does not target specific cell types, as is achievable with optogenetics. This noninvasive approach to driving 40-Hz neural activity should enable further research into the interactions between neural activity, molecular pathology, and the brain's immune system. Construction of the light-flicker system requires ~1 d and some electronics experience or available guidance. The flicker manipulation and assessment can be completed in a few days, depending on the experimental design.

Octaarginine-modified gold nanoparticles enhance the radiosensitivity of human colorectal cancer cell line LS180 to megavoltage radiation.

BACKGROUND: This study investigated the effectiveness and underpinning mechanisms of radiosensitization using octaarginine (R8)-modified gold nanoparticle-poly(ethylene glycol) (GNP-PEG-R8) in colorectal cancer cell line LS180 to megavoltage radiotherapy in vitro. METHOD: In-house synthesized GNP-PEG was characterized by transmission electron microscopy, dynamic light scattering, ultraviolet-visible spectrophotometry, and X-ray photoelectron spectroscopy. Inductively coupled plasma mass spectroscopy was used to quantify internalization. Direct cytotoxicity was established using the Cell Counting Kit-8, while radiosensitivity was determined using the gold standard in vitro clonogenic assay. Cell-cycle distribution, apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were analyzed by flow cytometry, further exploring the key mechanisms driving GNP-PEG-R8 radiosensitization. RESULTS: The core GNP diameter was 6.3±1.1 nm (mean±SD). Following functionalization, the hydrodynamic diameter increased to 19.7±2.8 nm and 27.8±1.8 nm for GNP-PEG and GNP-PEG-R8, with respective surface plasmon resonance peaks of 515 nm and 525 nm. Furthermore, incorporation of the R8 significantly increased nanoparticle internalization compared to GNP-PEG (p<0.001) over a 1 h treatment period. Functionalized GNPs confer little cytotoxicity below 400 nM. In clonogenic assays, radiation combined with GNP-PEG-R8 induced a significant reduction in colony formation compared with radiation alone, generating a sensitizer enhancement ratio of 1.59. Furthermore, GNP-PEG-R8 plus radiation predominantly induced cell-cycle arrest in the G2/M phase, increasing G2/M stalling by an additional 10% over GNP-PEG, markedly promoting apoptosis (p<0.001). Finally, ROS levels and alterations in MMP were investigated, indicating a highly significant (p<0.001) change in both parameters following the combined treatment of GNP-PEG-R8 and radiation over radiation alone. CONCLUSION: R8-modified GNPs were efficiently internalized by LS180 cells, exhibiting minimal cytotoxicity. This yielded significant radiosensitization in response to megavoltage radiation. GNP-PEG-R8 may enhance radiosensitivity by arresting cell cycle and inducing apoptosis, with elevated ROS identified as the likely initiator.

Near-infrared light-controlled regulation of intracellular calcium to modulate macrophage polarization.

Macrophages are multifunctional immune cells with diverse physiological functions such as fighting against infection, influencing progression of pathologies, maintaining homeostasis, and regenerating tissues. Macrophages can be induced to adopt distinct polarized phenotypes, such as classically activated pro-inflammatory (M1) phenotypes or alternatively activated anti-inflammatory and pro-healing (M2), to execute diverse and dynamic immune functions. However, unbalanced polarizations of macrophage can lead to various pathologies, such as atherosclerosis, obesity, tumor, and asthma. Thus, the capability to remotely control macrophage phenotypes is important to the success of treating many pathological conditions involving macrophages. In this study, we developed an upconversion nanoparticle (UCNP)-based photoresponsive nanocarrier for near-infrared (NIR) light-mediated control of intracellular calcium levels to regulate macrophage polarization. UCNP was coated with mesoporous silica (UCNP@mSiO2), into which loaded calcium regulators that can either supply or deplete calcium ions. UCNP@mSiO2 was chemically modified through serial coupling of photocleavable linker and Arg-Gly-Asp (RGD) peptide-bearing molecular cap via cyclodextrin-adamantine host-guest complexation. The RGD-bearing cap functioned as the photolabile gating structure to control the release of calcium regulators and facilitated the cellular uptake of UCNP@mSiO2 nanocarrier. The upconverted UV light emission from the UCNP@mSiO2 under NIR light excitation triggered the cleavage of cap and intracellular release of calcium regulators, thereby allowing temporal regulation on the intracellular calcium levels. Application of NIR light through skin tissue promoted M1 or M2 polarization of macrophages, by elevating or depleting intracellular calcium levels, respectively. To the best of our knowledge, this is the first demonstration of NIR light-mediated remote control on macrophage polarization. This photoresponsive nanocarrier offers the potential to remotely manipulate in vivo immune functions, such as inflammation or tissue regeneration, via NIR light-controlled macrophage polarization.

Effects of 12C6+ Heavy Ion Radiation on Dendritic Cells Function.

BACKGROUND Carbon ion radiotherapy has been shown to be more effective in cancer radiotherapy than photon irradiation. Influence of carbon ion radiation on cancer microenvironment is very important for the outcomes of radiotherapy. Tumor-infiltrating dendritic cells (DCs) play critical roles in cancer antigen processing and antitumor immunity. However, there is scant literature covering the effects of carbon ion radiation on DCs. In this study, we aimed to uncover the impact of carbon ion irradiation on bone marrow derived DCs. MATERIAL AND METHODS Bone marrow cells were co-cultured with GM-CSF and IL-4 for seven days, and the population of DCs was confirmed with flow cytometry. We used an Annexin V and PI staining method to detect cell apoptosis. Endocytosis assay of DCs was determined by using a flow cytometry method. DCs migration capacity was tested by a Transwell method. We also used ELISA assay and western blotting assay to examine the cytokines and protein expression, respectively. RESULTS Our data showed that carbon ion radiation induced apoptosis in both immature and mature DCs. After irradiation, the endocytosis and migration capacity of DCs was also impaired. Interestingly, carbon irradiation triggered a burst of IFN-g and IL-12 in LPS or CpG treated DCs, which provide novel insights into the combination of immunotherapy and carbon ion radiotherapy. Finally, we found that carbon ion irradiation induced apoptosis and migration suppression was p38 dependent. CONCLUSIONS Our present study demonstrated that carbon ion irradiation induced apoptosis in DCs, and impaired DCs function mainly through the p38 signaling pathway. Carbon ion irradiation also triggered anti-tumor cytokines secretion. This work provides novel information of carbon ion radiotherapy in DCs, and also provides new insights on the combination of immune adjuvant and carbon ion radiotherapy.

Unique Roles of ß-Arrestin in GPCR Trafficking Revealed by Photoinducible Dimerizers.

Intracellular trafficking of G protein-coupled receptors (GPCRs) controls their localization and degradation, which affects a cell's ability to adapt to extracellular stimuli. Although the perturbation of trafficking induces important diseases, these trafficking mechanisms are poorly understood. Herein, we demonstrate an optogenetic method using an optical dimerizer, cryptochrome (CRY) and its partner protein (CIB), to analyze the trafficking mechanisms of GPCRs and their regulatory proteins. Temporally controlling the interaction between ß-arrestin and ß2-adrenergic receptor (ADRB2) reveals that the duration of the ß-arrestin-ADRB2 interaction determines the trafficking pathway of ADRB2. Remarkably, the phosphorylation of ADRB2 by G protein-coupled receptor kinases is unnecessary to trigger clathrin-mediated endocytosis, and ß-arrestin interacting with unphosphorylated ADRB2 fails to activate mitogen-activated protein kinase (MAPK) signaling, in contrast to the ADRB2 agonist isoproterenol. Temporal control of ß-arrestin-GPCR interactions will enable the investigation of the unique roles of ß-arrestin and the mechanism by which it regulates ß-arrestin-specific trafficking pathways of different GPCRs.

Unraveling the cell-type dependent radiosensitizing effects of gold through the development of a multifunctional gold nanoparticle.

The radiosensitizing efficacy of gold is well established, however, there remain several significant barriers to the successful clinical translation of nano-sized gold particles (AuNPs). These barriers include: retaining stability in relevant biological sera, demonstrating effectiveness at clinically relevant AuNP concentrations and identifying the biological context where significant benefit is most likely to be achieved. Herein we have developed a AuNP preparation, stress-tested to provide effective protection from salt and serum mediated agglomeration. Furthermore, the core AuNP is co-functionalized with two biologically derived peptides designed to enhance endocytosis and promote endosomal escape, thus maximizing intracellular AuNP surface area. In summary, these investigations demonstrate restored AuNP internalization using the co-functionalized preparation that generated significant radiosensitization, in both in vitro and in vivo models, at clinically viable treatment concentrations. Furthermore, we have identified an underpinning biological mechanism in the inherent radical scavenging capacity that could be used to predict radiosensitizing efficacy.

Low-intensity pulsed ultrasound promotes cell motility through vinculin-controlled Rac1 GTPase activity.

Low-intensity pulsed ultrasound (LIPUS) is a therapy used clinically to promote healing. Using live-cell imaging we show that LIPUS stimulation, acting through integrin-mediated cell-matrix adhesions, rapidly induces Rac1 activation associated with dramatic actin cytoskeleton rearrangements. Our study demonstrates that the mechanosensitive focal adhesion (FA) protein vinculin, and both focal adhesion kinase (FAK, also known as PTK2) and Rab5 (both the Rab5a and Rab5b isoforms) have key roles in regulating these effects. Inhibiting the link of vinculin to the actin-cytoskeleton abolished LIPUS sensing. We show that this vinculin-mediated link was not only critical for Rac1 induction and actin rearrangements, but was also important for the induction of a Rab5-dependent increase in the number of early endosomes. Expression of dominant-negative Rab5, or inhibition of endocytosis with dynasore, also blocked LIPUS-induced Rac1 signalling events. Taken together, our data show that LIPUS is sensed by cell matrix adhesions through vinculin, which in turn modulates a Rab5-Rac1 pathway to control ultrasound-mediated endocytosis and cell motility. Finally, we demonstrate that a similar FAK-Rab5-Rac1 pathway acts to control cell spreading upon fibronectin.

Stress-specific p38 MAPK activation is sufficient to drive EGFR endocytosis but not its nuclear translocation.

EGF receptor (EGFR) endocytosis is induced by stress in a manner dependent on the p38 MAPK family. Ligand and stresses such as X-rays, reportedly promote nuclear trafficking of endocytosed EGFR for regulation of gene transcription and DNA repair. We fail to detect EGFR endocytosis or nuclear transport following X-ray treatment of HeLa or head and neck cancer cells, despite extensive DNA damage induction. Apparent nuclear staining with EGFR extracellular domain antibody remained present despite reduced/absent EGFR expression, and so did not represent nuclear EGFR. UVB and UVC, but not X-ray or UVA, treatment induced p38 activation and EGFR endocytosis, although all of these stresses induced DNA damage, indicating that DNA damage alone is not sufficient to induce EGFR endocytosis. Increased reactive oxygen species (ROS) levels following UVB treatment, compared to that seen with X-rays, do not alone explain differences in p38 activation. UVB, like UVC, induced EGFR accumulation predominantly in perinuclear endosomes, rather than in the nucleus. Our morphological techniques identifying major changes in receptor distribution do not exclude the possibility that small but biologically relevant amounts of EGFR enter the nucleus. This study highlights the importance and limitations of morphological analyses of receptor distribution in understanding signaling outcome.

Quantitative Analysis of Endocytic Recycling of Membrane Proteins by Monoclonal Antibody-Based Recycling Assays.

In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols. J. Cell. Physiol. 232: 463-476, 2017. © 2016 Wiley Periodicals, Inc.

Clathrin regulates blue light-triggered lateral auxin distribution and hypocotyl phototropism in Arabidopsis.

Phototropism is the process by which plants grow towards light in order to maximize the capture of light for photosynthesis, which is particularly important for germinating seedlings. In Arabidopsis, hypocotyl phototropism is predominantly triggered by blue light (BL), which has a profound effect on the establishment of asymmetric auxin distribution, essential for hypocotyl phototropism. Two auxin efflux transporters ATP-binding cassette B19 (ABCB19) and PIN-formed 3 (PIN3) are known to mediate the effect of BL on auxin distribution in the hypocotyl, but the details for how BL triggers PIN3 lateralization remain poorly understood. Here, we report a critical role for clathrin in BL-triggered, PIN3-mediated asymmetric auxin distribution in hypocotyl phototropism. We show that unilateral BL induces relocalization of clathrin in the hypocotyl. Loss of clathrin light chain 2 (CLC2) and CLC3 affects endocytosis and lateral distribution of PIN3 thereby impairing BL-triggered establishment of asymmetric auxin distribution and consequently, phototropic bending. Conversely, auxin efflux inhibitors N-1-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid affect BL-induced relocalization of clathrin, endocytosis and lateralization of PIN3 as well as asymmetric distribution of auxin. These results together demonstrate an important interplay between auxin and clathrin function that dynamically regulates BL-triggered hypocotyl phototropism in Arabidopsis.