Live-cell Fluorescence Microscopy Research Articles

Pathogenic LRRK2 regulates ciliation probability upstream of tau tubulin kinase 2 via Rab10 and RILPL1 proteins

Mutations that activate LRRK2 protein kinase cause Parkinson's disease. We showed previously that Rab10 phosphorylation by LRRK2 enhances its binding to RILPL1, and together, these proteins block cilia formation in a variety of cell types, including patient derived iPS cells. We have used live-cell fluorescence microscopy to identify, more precisely, the effect of LRRK2 kinase activity on both the formation of cilia triggered by serum starvation and the loss of cilia seen upon serum readdition. LRRK2 activity decreases the overall probability of ciliation without changing the rates of cilia formation in R1441C LRRK2 MEF cells. Cilia loss in these cells is accompanied by ciliary decapitation, and kinase activity does not change the timing or frequency of decapitation or the rate of cilia loss but increases the percent of cilia that are lost upon serum addition. LRRK2 activity, or overexpression of RILPL1 protein, blocks release of CP110 from the mother centriole, a step normally required for early ciliogenesis; LRRK2 blockade of CP110 uncapping requires Rab10 and RILPL1 proteins and is due to failure to recruit TTBK2, a kinase needed for CP110 release. In contrast, deciliation probability does not change in cells lacking Rab10 or RILPL1 and relies on a distinct LRRK2 pathway. These experiments provide critical detail to our understanding of the cellular consequences of pathogenic LRRK2 mutation and indicate that LRRK2 blocks ciliogenesis upstream of TTBK2 and enhances the deciliation process in response to serum addition.

Combining cross-coupling reaction and Knoevenagel condensation in the synthesis of glyco-BODIPY probes for DC-SIGN super-resolution bioimaging

Lectins are involved in a wide range of carbohydrate mediated recognition processes. Therefore, the availability of highly performant fluorescent tools tailored for lectin targeting and able to efficiently track events related to such key targets is in high demand. We report here on the synthesis of the glyco-BODIPYs 1 and 2, based on the efficient combination of a Heck-like cross coupling and a Knoevenagel condensation, which revealed efficient in addressing lectins. In particular, glyco-BODIPY 1 has two glycosidase stable C-mannose residues, which act as DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) targeting modules. By using live-cell fluorescence microscopy, we proved that BODIPY-mannose 1 was efficiently taken up by immune cells expressing DC-SIGN receptors. Super-resolution stimulated emission depletion (STED) microscopy further revealed that the internalized 1 localized in membranes of endosomes, proving that 1 is a reliable tool also in STED applications. Of note, glyco-BODIPY 1 contains an aryl-azido group, which allows further functionalization of the glycoprobe with bioactive molecules, thus paving the way for the use of 1 for tracking lectin-mediated cell internalization in diverse biological settings.

Curcumin Encapsulated in Crosslinked Cyclodextrin Nanoparticles Enables Immediate Inhibition of Cell Growth and Efficient Killing of Cancer Cells.

The efficiency of anti-cancer drugs is commonly determined by endpoint assays after extended incubation times, often after days. Here we demonstrate that curcumin encapsulated in crosslinked cyclodextrin nanoparticles (CD-NP) acts extremely rapidly on cell metabolism resulting in an immediate and complete inhibition of cell growth and in efficient cancer-cell killing only few hours after incubation. This early onset of anti-cancer action was discovered by live-cell high-throughput fluorescence microscopy using an environmental stage. To date, only very few examples of covalently crosslinked nanoscale CD-based (CD-NP) drug carriers exist. Crosslinking cyclodextrins enables the adsorption of unusually high payloads of hydrophobic curcumin (762 µg CC/mg CD-NP) reflecting a molar ratio of 2.3:1 curcumin to cyclodextrin. We have investigated the effect of CD-NP encapsulated curcumin (CD-CC-NP) in comparison to free, DMSO-derived curcumin nanoparticles (CC-NP) on 4 different cell lines. Very short incubations times as low as 1 h were applied and cell responses after medium change were subsequently followed over two days. We show that cell proliferation is inhibited nearly immediately in all cell lines and that a cell- and concentration dependent cancer-cell killing occurs. Anti-cancer effects were similar with free and encapsulated curcumin, however, encapsulation in CD-NP drastically extends the long-term photostability and anti-cancer activity of curcumin. Curcumin-sensitivity is highest in HeLa cells reaching up to 90% cell death under these conditions. Sensitivity decreased from HeLa to T24 to MDA MB-231 cells. Strikingly, the immortalized non-cancerous cell line MCF-10A was robust against curcumin concentrations that were highly toxic to the other cell lines. Our results underline the potential of curcumin as gentle and yet effective natural anti-cancer agent when delivered solvent-free in stabilizing and biocompatible drug carriers such as CD-NP that enable efficient cellular delivery.

Single-molecule fluorescence microscopy reveals modulation of DNA polymerase IV-binding lifetimes by UmuD (K97A) and UmuD'.

DNA polymerase IV (pol IV) is expressed at increased levels in Escherichia coli cells that suffer DNA damage. In a recent live-cell single-molecule fluorescence microscopy study, we demonstrated that the formation of pol IV foci is strongly recB-dependent in cells treated with the DNA break-inducing antibiotic ciprofloxacin. The results of that study support a model in which pol IV acts to extend D-loop structures during recombinational repair of DNA double-strand breaks. In the present study, we extend upon this work, investigating the UmuD and UmuD' proteins as potential modulators of pol IV activity in ciprofloxacin-treated cells. We found that the non-cleavable mutant UmuD(K97A) promotes long-lived association of pol IV with the nucleoid, whereas its cleaved form, UmuD', which accumulates in DNA-damaged cells, reduces binding. The results provide additional support for a model in which UmuD and UmuD' directly modulate pol IV-binding to the nucleoid.

The intracellular bacterium Rickettsia rickettsii exerts an inhibitory effect on the apoptosis of tick cells

BackgroundRickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain spotted fever, a life-threatening illness. To obtain an insight into the vector–pathogen interactions, we assessed the effects of infection with R. rickettsii on the proteome cells of the tick embryonic cell line BME26.MethodsThe proteome of BME26 cells was determined by label-free high-performance liquid chromatography coupled with tandem mass spectrometry analysis. Also evaluated were the effects of infection on the activity of caspase-3, assessed by the hydrolysis of a synthetic fluorogenic substrate in enzymatic assays, and on the exposition of phosphatidyserine, evaluated by live-cell fluorescence microscopy after labeling with annexin-V. Finally, the effects of activation or inhibition of caspase-3 activity on the growth of R. rickettsii in BME26 cells was determined.ResultsTick proteins of different functional classes were modulated in a time-dependent manner by R. rickettsii infection. Regarding proteins involved in apoptosis, certain negative regulators were downregulated at the initial phase of the infection (6 h) but upregulated in the middle of the exponential phase of the bacterial growth (48 h). Microorganisms are known to be able to inhibit apoptosis of the host cell to ensure their survival and proliferation. We therefore evaluated the effects of infection on classic features of apoptotic cells and observed DNA fragmentation exclusively in noninfected cells. Moreover, both caspase-3 activity and phosphatidylserine exposition were lower in infected than in noninfected cells. Importantly, while the activation of caspase-3 exerted a detrimental effect on rickettsial proliferation, its inhibition increased bacterial growth.ConclusionsTaken together, these results show that R. rickettsii modulates the proteome and exerts an inhibitory effect on apoptosis in tick cellsthat seems to be important to ensure cell colonization.Graphical

The Performance of Minicircle DNA Versus Parental Plasmid in p53 Gene Delivery Into HPV-18-Infected Cervical Cancer Cells

Minicircle DNA (mcDNA) has been suggested as a vanguard technology for gene therapy, consisting of a nonviral DNA vector devoid of prokaryotic sequences. Unlike conventional plasmid DNA (pDNA), this small vector is able to sustain high expression rates throughout time. Thus, this work describes the construction, production, and purification of mcDNA-p53 and its precursor parental plasmid (PP)-p53 for a comparative study of both DNA vectors in the growth suppression of human papillomavirus (HPV)-18-infected cervical cancer cells. First, live cell imaging and fluorescence microscopy studies allowed to understand that mcDNA-p53 vector was able to enter cell nuclei more rapidly than PP-p53 vector, leading to a transfection efficiency of 68% against 34%, respectively. Then, p53 transcripts and protein expression assessment revealed that both vectors were able to induce transcription and the target protein expression. However, the mcDNA-p53 vector performance stood out, by demonstrating higher p53 expression levels (91.65 ± 2.82 U/mL vs. 74.75 ± 4.44 U/mL). After assuring the safety of both vectors by viability studies, such potential was confirmed by proliferation and apoptosis assays. These studies confirmed the mcDNA-p53 vector function toward cell cycle arrest and apoptosis in HPV-18-infected cervical cancer cells. Altogether, these results suggest that the mcDNA vector has a more promising and efficient role as a DNA vector than conventional pDNA, opening new investigation lines for cervical cancer treatment in the future.

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin.

SummaryEstrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

Live Cell Microscopy of Murine Polyomavirus Subnuclear Replication Centers.

During polyomavirus (PyV) infection, host proteins localize to subnuclear domains, termed viral replication centers (VRCs), to mediate viral genome replication. Although the protein composition and spatial organization of VRCs have been described using high-resolution immunofluorescence microscopy, little is known about the temporal dynamics of VRC formation over the course of infection. We used live cell fluorescence microscopy to analyze VRC formation during murine PyV (MuPyV) infection of a mouse fibroblast cell line that constitutively expresses a GFP-tagged replication protein A complex subunit (GFP-RPA32). The RPA complex forms a heterotrimer (RPA70/32/14) that regulates cellular DNA replication and repair and is a known VRC component. We validated previous observations that GFP-RPA32 relocalized to sites of cellular DNA damage in uninfected cells and to VRCs in MuPyV-infected cells. We then used GFP-RPA32 as a marker of VRC formation and expansion during live cell microscopy of infected cells. VRC formation occurred at variable times post-infection, but the rate of VRC expansion was similar between cells. Additionally, we found that the early viral protein, small TAg (ST), was required for VRC expansion but not VRC formation, consistent with the role of ST in promoting efficient vDNA replication. These results demonstrate the dynamic nature of VRCs over the course of infection and establish an approach for analyzing viral replication in live cells.

Live-SIMBA: an ImageJ plug-in for the universal and accelerated single molecule-guided Bayesian localization super resolution microscopy (SIMBA) method.

Live-cell super-resolution fluorescence microscopy techniques allow biologists to observe subcellular structures, interactions and dynamics at the nanoscale level. Among of them, single molecule-guided Bayesian localization super resolution microscopy (SIMBA) and its derivatives produce an appropriate 50 nm spatial resolution and a 0.1-2s temporal resolution in living cells with simple off-the-shelf total internal reflection fluorescence (TIRF) equipment. However, SIMBA and its derivatives are limited by the requirement for dual-channel dataset or single-channel dataset with special design, the time-consuming calculation for extended field of view and the lack of real-time visualization tool. Here, we propose a universal and accelerated SIMBA ImageJ plug-in, Live-SIMBA, for time-series analysis in living cells. Live-SIMBA circumvents the requirement of dual-channel dataset using intensity-based sampling algorithm and improves the computing speed using multi-core parallel computing technique. Live-SIMBA also better resolves the weak signals inside the specimens with adjustable background estimation and distance-threshold filter. With improved fidelity on reconstructed structures, greatly accelerated computation, and real-time visualization, Live-SIMBA demonstrates its extended capabilities in live-cell super-resolution imaging.

Nanotopography Enhances Dynamic Remodeling of Tight Junction Proteins through Cytosolic Liquid Complexes.

Nanotopographic materials provide special biophysical stimuli that can regulate epithelial tight junctions and their barrier function. Through the use of total internal reflection fluorescence microscopy of live cells, we demonstrated that contact of synthetic surfaces with defined nanotopography at the apical surface of epithelial monolayers increased paracellular permeability of macromolecules. To monitor changes in tight junction morphology in live cells, we fluorescently tagged the scaffold protein zonula occludens-1 (ZO-1) through CRISPR/Cas9-based gene editing to enable live cell tracking of ZO-1 expressed at physiologic levels. Contact between cells and nanostructured surfaces destabilized junction-associated ZO-1 and promoted its arrangement into highly dynamic liquid cytosolic complexes with a 1-5 μm diameter. Junction-associated ZO-1 rapidly remodeled, and we observed the direct transformation of cytosolic complexes into junction-like structures. Claudin-family tight junction transmembrane proteins and F-actin also were associated with these ZO-1 containing cytosolic complexes. These data suggest that these cytosolic structures are important intermediates formed in response to nanotopographic cues that facilitate rapid tight junction remodeling in order to regulate paracellular permeability.

Bioaerosol sampling of patients with suspected pulmonary tuberculosis: a study protocol

BackgroundTuberculosis (TB) is transmitted in bioaerosols containing Mycobacterium tuberculosis (Mtb). Despite being central to ongoing TB transmission, no routine diagnostic assay exists to measure Mtb in bioaerosols. Furthermore, published studies of Mtb in bioaerosol samples have been limited to individuals with sputum-positive pulmonary TB. Notably, TB diagnosis is based on clinical symptoms and sputum laboratory findings. This is despite the fact that approximately half of all patients commencing TB treatment are sputum-negative, resulting in a high proportion of presumptive treatments. Here, we propose to use a sensitive air sampling protocol to investigate the prevalence of Mtb-containing bioaerosols in both sputum-positive and sputum-negative TB suspects, at the same time evaluating the potential to identify unrecognized transmitters of TB.MethodsOur parallel-group design will identify viable Mtb in bioaerosols produced by individuals attending a TB clinic in South Africa. Sampling will be performed on eligible individuals presenting with symptoms indicative of TB and repeated at 14 days if initially positive. Participants will be prospectively classified into three distinct groups based on National TB Control Program (NTBCP) criteria: Group A, TB notification with sputum-based laboratory confirmation; Group B, TB notification with empiric diagnosis; and Group C, individuals not notified. Group C individuals with detectable Mtb bioaerosol will be monitored until resolution of clinical and laboratory status. Collection of bioaerosol specimens will be via two consecutive sampling modalities: (1) direct sampling following a specific respiratory manoeuvre; and (2) indirect sampling during passive respiratory activity. Bioaerosol specimens will be analyzed for viable Mtb using DMN-trehalose staining and live-cell fluorescence microscopy. Mtb genomes and mycobacterial and host lipids will be detected using droplet digital PCR and mass spectrometry analyses, respectively. The primary objective is to determine the prevalence of Mtb bioaerosols in all TB clinic attendees and in each of the groups. Secondary objectives are to investigate differences in prevalence of Mtb bioaerosol by HIV status and current isoniazid preventive therapy (IPT) use; we will also determine the impact of anti-TB chemotherapy on Mtb-containing bioaerosol production.DiscussionRespiratory bioaerosol has a potential role in non-invasive TB diagnosis, infectivity measurement and treatment monitoring.Trial registrationClinicalTrials.gov: NCT04241809. Date of Registration: 27/1/2020.

Orthogonal fluorescent chemogenetic reporters for multicolor imaging.

Spectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. Here, we present the engineering of a set of spectrally orthogonal fluorogen activating tags based on the Fluorescence Activating and absorption Shifting Tag (FAST), that are compatible with two-color, live cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development, and the development of split complementation systems capable of detecting multiple protein-protein interactions by live cell fluorescence microscopy.

Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis.

Clathrin-mediated endocytosis (CME) is a crucial cellular process implicated in many aspects of plant growth, development, intra- and intercellular signaling, nutrient uptake and pathogen defense. Despite these significant roles, little is known about the precise molecular details of how CME functions in planta To facilitate the direct quantitative study of plant CME, we review current routinely used methods and present refined, standardized quantitative imaging protocols that allow the detailed characterization of CME at multiple scales in plant tissues. These protocols include: (1) an efficient electron microscopy protocol for the imaging of Arabidopsis CME vesicles in situ, thus providing a method for the detailed characterization of the ultrastructure of clathrin-coated vesicles; (2) a detailed protocol and analysis for quantitative live-cell fluorescence microscopy to precisely examine the temporal interplay of endocytosis components during single CME events; (3) a semi-automated analysis to allow the quantitative characterization of global internalization of cargos in whole plant tissues; and (4) an overview and validation of useful genetic and pharmacological tools to interrogate the molecular mechanisms and function of CME in intact plant samples.This article has an associated First Person interview with the first author of the paper.

Autophagy induction by exogenous polyamines is an artifact of bovine serum amine oxidase activity in culture serum

Polyamines are small polycationic alkylamines involved in many fundamental cellular processes, including proliferation, nucleic acid synthesis, apoptosis, and protection from oxidative damage. It has been proposed that in addition to these functions, elevated levels of polyamines promote longevity in various biological systems, including yeast, Drosophila, and murine models. A series of in vitro mechanistic studies by multiple investigators has led to the conclusion that addition of exogenous spermidine promotes longevity through autophagy induction; however, these experiments were confounded by the use of mammalian cell culture systems supplemented with fetal bovine serum. Using cell viability assays, LC3B immunoblots, and live-cell fluorescence microscopy, we report here that in the presence of ruminant serum, exogenously added polyamines are quickly oxidized by the copper-containing bovine serum amine oxidase. This polyamine oxidation resulted in the production of harmful byproducts including hydrogen peroxide, ammonia, and reactive aldehydes. Our data demonstrate that it is critically important to prevent confounding bovine serum amine oxidase-induced cytotoxicity in mechanistic studies of the roles of polyamines in autophagy.

Polymersome Formation by Amphiphilic Polyglycerol-b-polydisulfide-b-polyglycerol and Glutathione-Triggered Intracellular Drug Delivery.

This article reports the synthesis, spontaneous self-assembly, highly efficient drug encapsulation, and glutathione (GSH)-triggered intracellular sustained drug delivery of an ABA-type amphiphilic triblock copolymer, namely, polyglycerol-b-poly(disulfide)-b-polyglycerol (PG-b-PDS-b-PG). The bioreducible PDS block with reactive pyridyldisulfide groups present at the chain terminals was attached to thiol-terminated heterotelechelic PG by a thiol-disulfide exchange reaction producing the amphiphilic PG-b-PDS-b-PG. It formed a stable polymersome in aqueous medium with a critical aggregation concentration of 0.02 mg/mL and average hydrodynamic diameter (Dh) of 230 nm and showed highly efficient and stable encapsulation of doxorubicin (Dox) with a remarkably high drug loading efficiency (DLE) and drug loading content (DLC) of 54% and 16%, respectively. Fluorescence spectroscopy studies revealed GSH-triggered drug release and strong dependence of the release kinetics on the GSH concentration due to degradation of the amphiphilic block copolymer and disassembly of the polymersome. MTT assay indicated excellent biocompatibility of the block copolymer as >90% cells (HeLa or MDA-MB-231) were found to be alive after 96 h of incubation with a polymer concentration of up to 1.0 mg/mL, which was further validated by the hemolysis assay. Cytotoxicity assay of the Dox-loaded polymersome exhibited time and dose-dependent sustained killing of HeLa as well as MDA-MB-231 cells wherein after 48 h of incubation >50% cell killing was noticed with a Dox concentration of ∼4.0 and ∼8.7 μg/mL, respectively, while the free Dox showed faster cell killing. Flow cytometry and live cell fluorescence microscopy studies revealed time-dependent cellular uptake of the drug-loaded polymersome followed by diffusion of the drug to the nucleus. Cells with artificially enhanced GSH were killed at a much faster rate indicating that intracellular GSH-triggered disassembly is the key drug release mechanism.

A microscopy-based kinetic analysis of yeast vacuolar protein sorting.

Saccharomyces cerevisiae is amenable to studying membrane traffic by live-cell fluorescence microscopy. We used this system to explore two aspects of cargo protein traffic through prevacuolar endosome (PVE) compartments to the vacuole. First, at what point during Golgi maturation does a biosynthetic vacuolar cargo depart from the maturing cisternae? To address this question, we modified a regulatable fluorescent secretory cargo by adding a vacuolar targeting signal. Traffic of the vacuolar cargo requires the GGA clathrin adaptors, which arrive during the early-to-late Golgi transition. Accordingly, the vacuolar cargo begins to exit the Golgi near the midpoint of maturation, significantly before exit of a secretory cargo. Second, how are cargoes delivered from PVE compartments to the vacuole? To address this question, we tracked biosynthetic and endocytic cargoes after they had accumulated in PVE compartments. The results suggest that stable PVE compartments repeatedly deliver material to the vacuole by a kiss-and-run mechanism.

Synthesis of a far-red emitting flavonoid-based lysosome marker for live cell imaging applications

A bright far-red emitting flavonoid derivative (FuraET) was synthesized in good yields by inserting a π extension group (i.e., furan) into the flavonoid skeleton, via using the Suzuki-Miyaura cross-coupling reaction. FuaraET exhibited optical absorption at λab ≈ 450 nm and emission λem ≈ 660 nm by recognizing as the first far-red emitting flavonoid derivative reported. FuraET exhibited a large Stokes shift (Δλ > 150 nm) high fluorescent quantum yield (φfl ≈ 0.2–0.4), and good photostability indicating excellent characteristics for an imaging probe. Live cell fluorescent confocal microscopy imaging revealed the exceptional selectivity of the FuraET towards cellular lysosomes (Mander’s overlap coefficients >0.9). The observed non-alkalinizing nature and high biocompatibility (LC50 > 50 µM) suggested that FuraET can a reliable lysosome marker for live cell imaging experiments. Our further study also indicated that FuraET may likely internalized into hydrophobic regions of the cellular lysosomes in contrast to acidic lysosomal lumen.

The HSV-1 ICP4 Transcriptional Auto-Repression Circuit Functions as a Transcriptional "Accelerator" Circuit.

Herpes simplex virus-1 (HSV-1) is a significant human pathogen. Upon infection, HSV-1 expresses its immediate early (IE) genes, and the IE transcription factor ICP4 (infectious cell protein-4) plays a pivotal role in initiating the downstream gene-expression cascade. Using live-cell time-lapse fluorescence microscopy, flow cytometry, qPCR, and chromatin immunoprecipitation, we quantitatively monitored the expression of ICP4 in individual cells after infection. We find that extrinsic stimuli can accelerate ICP4 kinetics without increasing ICP4 protein or mRNA levels. The accelerated ICP4 kinetics—despite unchanged steady-state ICP4 protein or mRNA level—correlate with increased HSV-1 replicative fitness. Hence, the kinetics of ICP4 functionally mirror the kinetics of the human herpesvirus cytomegalovirus IE2 “accelerator” circuit, indicating that IE accelerator circuitry is shared among the alpha and beta herpesviruses. We speculate that this circuit motif is a common evolutionary countermeasure to throttle IE expression and thereby minimize the inherent cytotoxicity of these obligate viral transactivators.

Neuronal contact guidance and YAP signaling on ultra-small nanogratings

Contact interaction of neuronal cells with extracellular nanometric features can be exploited to investigate and modulate cellular responses. By exploiting nanogratings (NGs) with linewidth from 500 nm down to 100 nm, we here study neurite contact guidance along ultra-small directional topographies. The impact of NG lateral dimension on the neuronal morphotype, neurite alignment, focal adhesion (FA) development and YAP activation is investigated in nerve growth factor (NGF)-differentiating PC12 cells and in primary hippocampal neurons, by confocal and live-cell total internal reflection fluorescence (TIRF) microscopy, and at molecular level. We demonstrate that loss of neurite guidance occurs in NGs with periodicity below 400 nm and correlates with a loss of FA lateral constriction and spatial organization. We found that YAP intracellular localization is modulated by the presence of NGs, but it is not sensitive to their periodicity. Nocodazole, a drug that can increase cell contractility, is finally tested for rescuing neurite alignment showing mild ameliorative effects. Our results provide new indications for a rational design of biocompatible scaffolds for enhancing nerve-regeneration processes.

Surface Activity and Lipid Droplet Localization of Full Length and Truncated Perilipin 3

Lipid droplets (LDs) are ubiquitously expressed organelles that are critical for cellular energy regulation. LDs consist of a neutral lipid core and a phospholipid monolayer, which allows for protein localization and binding. The mechanism of protein-lipid interactions at the LD interface remain poorly understood, especially when compared to those that occur at biological membranes. In order to better understand protein-lipid interactions at the LD monolayer we focus on LD binding proteins of the perilipin family. The perilipins are a family of LD binding proteins that are under increased scrutiny due to their abundance in mammalian cells. Perilipin 3, a member of this family, contains a conserved bundle of amphipathic α-helices located at its C-terminus. This α-helix bundle is noteworthy due to its resemblance to the lipid binding domains of the well characterized apolipoproteins, apoE and apoLpIII. Previous data from our lab suggest that this C-terminal α-helix bundle on perilipin 3 has a stronger interaction with the LD monolayer than the full-length protein. To obtain a more comprehensive picture of the function of the C-terminal domain, we compare the full-length perilipin 3 to a truncated sequence that only contains the C-terminal domain, using pendant drop tensiometry to examine their binding to simulated LDs with lipid monolayers containing different acyl-chain compositions and headgroups. We also use live-cell fluorescence microscopy with a small tetracysteine tag in order to determine the localization of full-length perilipin 3 versus the C-terminal truncation in murine adipocytes.