An Investigation of Hair and its Keratin Associated Proteins using Advanced Light Microscopy

Quantification and understanding of hair damage are often derived using semi-quantitative means which provide limited information regarding the whole hair. Furthermore, the roles of the varied keratin associated proteins (KAPs), which are the second most abundant proteins within hair, are poorly understood. Finally, the movement of compounds into the hair fibre has never previously been analysed dynamically or using the range of techniques currently available. Fluorescence lifetime imaging microscopy (FLIM) and correlative techniques are utilised to quantify and understand the chemical changes taking place following damage. Super-resolution imaging techniques, including Airyscan and 3D structured illumination microscopy (3D-SIM) are used for the imaging of a selection of fluorescently-tagged KAPs within transiently transfected HaCaT cells in order to describe their cell biology. Fluorescence recovery after photobleaching (FRAP) is also applied to gather information regarding the dynamics of these proteins. Additionally, Airyscan and FRAP are utilised for the analysis of fluorescent dye movement within hair to characterise dye pathways and partitioning in hair dynamically. FLIM provides a rapid and sensitive means to quantify oxidative damage to hair spatially, which is correlated to the conversion of tryptophan, through oxidation, to products including kynurenine. The uptake of dye into hair is dependent upon the size and lipophilicity of the molecule and may be limited due to a cuticle-cortex boundary observed. Knowledge of compound uptake into hair may be translated for the development of future cosmetics. The KAP subfamilies display distinct subcellular localisations to cytoskeletal components including epidermal keratins and actin structures. Functional characterisation of KAPs carried out in this study could allow a targeted approach to KAP-KAP and KAP-keratin interactions in future cosmetic treatments. This understanding may also be of relevance to the mechanism of certain diseases unrelated to the hair follicle, and indeed for the production of keratin-based biomaterials

Direct and dynamic detection of HIV-1 in living cells.

In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA) at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research

Multimodal Microscopy of Focal Adhesions

Through the systematic application of various advanced light microscopy techniques, including super-resolution microscopy, we have described in greater detail the dynamic structure of focal adhesions, protein complexes through which cells attach to a surface. We show that the dynamics of focal adhesion proteins correlate with the position and orientation of a focal adhesion within the cell, that focal adhesions around the actin fibre attachment site have a higher dens

Viral RNAs are unusually compact.

A majority of viruses are composed of long single-stranded genomic RNA molecules encapsulated by protein shells with diameters of just a few tens of nanometers. We examine the extent to which these viral RNAs have evolved to be physically compact molecules to facilitate encapsulation. Measurements of equal-length viral, non-viral, coding and non-coding RNAs show viral RNAs to have among the smallest sizes in solution, i.e., the highest gel-electrophoretic mobilities and the smallest hydrodynamic radii. Using graph-theoretical analyses we demonstrate that their sizes correlate with the compactness of branching patterns in predicted secondary structure ensembles. The density of branching is determined by the number and relative positions of 3-helix junctions, and is highly sensitive to the presence of rare higher-order junctions with 4 or more helices. Compact branching arises from a preponderance of base pairing between nucleotides close to each other in the primary sequence. The density of branching represents a degree of freedom optimized by viral RNA genomes in response to the evolutionary pressure to be packaged reliably. Several families of viruses are analyzed to delineate the effects of capsid geometry, size and charge stabilization on the selective pressure for RNA compactness. Compact branching has important implications for RNA folding and viral assembly

On the Digital Holographic Interferometry of Fibrous Material, I. Optical Properties of Polymer and Optical Fibers

The digital holographic interferometry (DHI) was utilized for investigating the optical properties of polymer and optical fibers. The samples investigated here were polyvinylidene fluoride (PVDF) polymer fiber and graded-index (GRIN) optical fiber. The phase shifting Mach-Zehnder interferometer was used to obtain five phase-shifted holograms, in which the phase difference between two successive holograms is pi/2, for each fiber sample. These holograms were recorded using a CCD camera and were combined to gain a complex wavefield, which was numerically reconstructed using the convolution approach into amplitude and phase distributions. The reconstructed phase distribution was used to determine the refractive index, birefringence and refractive index profile of the studied samples. The mean refractive index has been measured with accuracy up to 4 {\times} 10-4. The main advantage of DHI is to overcome the manual focusing limitations by means of the numerical focusing. The results showed accurate measurements of the optical properties of fibers.Comment: abstract, reference

Novel insights into hair structure and the effects of chemical stressors on hair and skin using label-free advanced light microscopy

There is a need for a better method to image hair as the current methods involve embedding the hair in resin, which may produce artefacts, or using dyes which are limited in their depth of penetration into the hair. The research performed in this thesis endeavours to characterise the cellular structure of human hair with label-free imaging using autofluorescence and fluorescence lifetime imaging. Wavelengths were shown to selectively excite the hair cuticle, cortex and medulla, and subcellular compartments. Development of an optical transverse imaging method enabled discoveries including different fluorescence lifetimes across the cuticle cell layers and suggests the cuticle layers possess differing chemical environments. A new method was developed to distinguish between eumelanin and pheomelanin using 405nm and 633nm wavelengths. The newly developed methods were additionally used in the characterisation of an unidentified hair and skin disorder, which found poorly differentiated cuticle cells and showed differences in the fluorescence lifetimes of the hair compared to control hairs. The hair care industry needs more efficacious chemical depilatories and information into their action. This was elucidated using the developed methods and a new dynamic imaging method. Potassium thioglycolate was shown to cause drastic expansion of the hair which was amplified by the addition of guanidine carbonate, creating fissures through the cuticle and into the cortex. Other experimental depilatory formulations were tested and were found to have varying effects upon the structure of the hair. New chemical depilatories require development because existing depilatories can cause irritation in the skin. Potassium thioglycolate and guanidine carbonate were tested on HaCaT cells, isolated cornified envelopes, and HEKn cells in a 3D epidermal model. An investigation into the differentiation, proliferation and acute stress response of the cells showed that the treatments had no significant effect on these markers. However, the chemicals negatively affected HaCaT cell viability and damaged the cornified envelopes. Despite this, the viability and structural integrity of the living cells of the epidermal model were maintained through the protection provided by the stratum corneum

Minihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overload

Iron overload is the hallmark of hereditary hemochromatosis and a complication of iron-loading anemias such as β-thalassemia. Treatment can be burdensome and have significant side effects, and new therapeutic options are needed. Iron overload in hereditary hemochromatosis and β-thalassemia intermedia is caused by hepcidin deficiency. Although transgenic hepcidin replacement in mouse models of these diseases prevents iron overload or decreases its potential toxicity, natural hepcidin is prohibitively expensive for human application and has unfavorable pharmacologic properties. Here, we report the rational design of hepcidin agonists based on the mutagenesis of hepcidin and the hepcidin-binding region of ferroportin and computer modeling of their docking. We identified specific hydrophobic/aromatic residues required for hepcidin-ferroportin binding and obtained evidence in vitro that a thiol-disulfide interaction between ferroportin C326 and the hepcidin disulfide cage may stabilize binding. Guided by this model, we showed that 7–9 N-terminal amino acids of hepcidin, including a single thiol cysteine, comprised the minimal structure that retained hepcidin activity, as shown by the induction of ferroportin degradation in reporter cells. Further modifications to increase resistance to proteolysis and oral bioavailability yielded minihepcidins that, after parenteral or oral administration to mice, lowered serum iron levels comparably to those after parenteral native hepcidin. Moreover, liver iron concentrations were lower in mice chronically treated with minihepcidins than those in mice treated with solvent alone. Minihepcidins may be useful for the treatment of iron overload disorders

The spectraplakin Dystonin antagonizes YAP activity and suppresses tumourigenesis

Aberrant expression of the Spectraplakin Dystonin (DST) has been observed in various cancers, including those of the breast. However, little is known about its role in carcinogenesis. In this report, we demonstrate that Dystonin is a candidate tumour suppressor in breast cancer and provide an underlying molecular mechanism. We show that in MCF10A cells, Dystonin is necessary to restrain cell growth, anchorage-independent growth, self-renewal properties and resistance to doxorubicin. Strikingly, while Dystonin maintains focal adhesion integrity, promotes cell spreading and cell-substratum adhesion, it prevents Zyxin accumulation, stabilizes LATS and restricts YAP activation. Moreover, treating DST-depleted MCF10A cells with the YAP inhibitor Verteporfin prevents their growth. In vivo, the Drosophila Dystonin Short stop also restricts tissue growth by limiting Yorkie activity. As the two Dystonin isoforms BPAG1eA and BPAG1e are necessary to inhibit the acquisition of transformed features and are both downregulated in breast tumour samples and in MCF10A cells with conditional induction of the Src proto-oncogene, they could function as the predominant Dystonin tumour suppressor variants in breast epithelial cells. Thus, their loss could deem as promising prognostic biomarkers for breast cancer.The authors acknowledge the support of the Bloomington Drosophila Stock Centre, the National Institute of Genetics (NIG-Fly) the services of the Animal, Imaging and Cytometry and Genomics facilities at Instituto Gulbenkian de Ciência, and of the i3S Scientific Platforms, including the Cell Culture and Genotyping (CCGen) and the Genomics (GenCore) platforms, as well as the Bioimaging and the Advanced Light Microscopy platforms. We are also grateful to M. J. Amorim and N. Tapon for reagents and to A. Monteiro for fly food preparation. We specially thank K. Struhl for providing the TAM-inducible ER-Src and PBabe cell lines and Rafeeq Mir, Eurico Morais-de-Sá, Archana Pawar and Carla Oliveira for comments on the manuscript. This work was supported by funds from Fundação para a Ciência e Tecnologia (FCT), co-financed by Fundo Europeu de Desenvolvimento Regional (FEDER) through Programa Operacional Competitividade e Internacionalização (POCI) (POCI-01-0145-FEDER-016390) and the Laço Grant in breast cancer 2015 to F.J. The i3S Bioimaging and Advanced Light Microscopy scientific platforms are both member of the national infrastructure PPBI-Portuguese Platform of BioImaging, supported by POCI-01-0145-FEDER-022122. P.J. was the recipient of fellowships from FCT (PD/ BD/52439/2013). F.J. was the recipient of IF/01031/2012