Investigation of age-related alterations occurring on the protein and the lipid level by using STED Microscopy

High resolution light microscopy is gaining in importance in the Life Sciences. This thesis reports on two applications of the STED (stimulated emission depletion) principle investigating alterations on the protein and lipid level occurring during aging. One hallmark of age-related neurodegenerative diseases is the abnormal post-translationally occurring hyperphosphorylation of proteins. Here, the interplay of two main post-translational modifications of neurofilaments, phosphorylation and glycosylation, was investigated. The organization pattern of these modifications can not be resolved by conventional diffraction-limited light microscopy. However, with the about 10-times higher resolution of STED microscopy the organization for both modifications was revealed. Long term glucose deprivation of redifferentiated neuroblastomas results in axonal swellings and cell death, which could not be rescued by refeeding glucose. These severe alterations could be observed by confocal resolution (~250 nm). Taking advantage of the higher resolution of the STED microscope (20-30 nm) allowed the detection of earlier alterations in the pattern of both modifications, where cell death could be prevented by the refeeding of glucose. Two kinases were found to be involved in the phosphorylation events occurring upon glucose deprivation in redifferentiated SH-SY5Y: the mitogen activated protein kinase p38 MAPK and to a lower extent also the stress activated protein kinase JNK. An effect occurring during aging is the change in the plasma membrane's fluidity. This thesis reports on the investigation of the diffusion characteristics of different phospholipids in dependency of the endogenous cholesterol content on a nanoscale. Fluorescent mono- and diacyl-phospholipid analogs were incorporated via BSA-complex formation into the plasma membrane of living cells. It was found that for best incorporation a molar ratio of 1:1 (fatty acid chains:BSA molecules) had to be used. Analogs of the phosphoglycerolipids and the sphingolipids, respectively, were the central study in this thesis. It was shown that sphingolipids, in contrast to phosphoglycerolipids, were trapped on spatial scales below 30 nm, which could not be examined by confocal microscopy. Upon changing the endogenous cholesterol level, enzymatically with cholesterol oxidase and by host-guest complex formation using the oligosaccharide β-cyclodextrin, respectively, this hindered-diffusion could be reversibly abolished

Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters

We demonstrate nanoscale resolution in far-field fluorescence microscopy using reversible photoswitching and localization of individual fluorophores at comparatively fast recording speeds and from the interior of intact cells. These advancements have become possible by asynchronously recording the photon bursts of individual molecular switching cycles. We present images from the microtubular network of an intact mammalian cell with a resolution of 40 nm

Photostable, Amino Reactive and Water-Soluble Fluorescent Labels Based on Sulfonated Rhodamine with a Rigidized Xanthene Fragment

Highly water soluble fluorescentdyes were synthesized and transformedinto new amino reactive fluorescentlabels for biological microscopy.To this end,rhodamine 8 (preparedfrom 7-hydroxy-1,2,3,4-tetrahydroquinoline(7) and phthalic anhydride in85% aq. H3PO4) was sulfonated with30% SO3 in H2SO4 and afforded thewater soluble disulfonic acid 3a (64%).Amidation of the carboxy group in 3awith 2-(methylamino)ethanol in thepresence of O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium·PF6(HATU) led to alcohol 3b (66%),which was transformed into the aminoreactive mixed carbonate 3d with di(Nsuccinimidyl)carbonate and Et3N. Reactionof the carboxy group in 3a withMeNHACHTUNGTRENUNG(CH2)2CO2Me and N,N,N’,N’-tetramethyl-O-(N-succinimidyl)-uronium·BF4 (TSTU) yielded methylester 13. After saponification of the aliphaticcarboxy group in 13,the compoundwas converted into NHS-ester3e (using HATU and Et3N). Heatingof 7 with trimellitic anhydride inH3PO4 gave a mixture of dicarboxylicacids 14 and 15 (1:1). Regioisomer 15was isolated,sulfonat ed with 30% SO3in H2SO4,and disulfonic acid 3 f wasused for the synthesis of the monoNHS-ester 3g,in which the stericallyunhindered carboxy group was selectivelyactivated (with N-hydroxysuccinimide,HA TU,and Et3N). The sulfonatedrhodamines 3b, c and f are solublein water (up to 0.1m),have excellentphotostabilities and large fluorescencequantum yields. Subdiffractionresolution images of tubulin filamentsof mammalian cells stained with thesedyes illustrate their applicability aslabels for stimulated emission depletionmicroscopy and other fluorescencetechniques.Fil: Boyarskiy, Vadim P.. Max Planck Institute for Biophysical Chemistry; Alemania. Saint Petersburg State University; RusiaFil: Belov, Vladimir N.. Max Planck Institute for Biophysical Chemistry; AlemaniaFil: Medda, Rebecca. Max Planck Institute for Biophysical Chemistry; AlemaniaFil: Hein, Birka. Max Planck Institute for Biophysical Chemistry; AlemaniaFil: Bossi, Mariano Luis. Max Planck Institute for Biophysical Chemistry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry; Alemani

Matrix-Immobilized BMP-2 on Microcontact Printed Fibronectin as an in vitro Tool to Study BMP-Mediated Signaling and Cell Migration

During development, growth factors (GFs) such as bone morphogenetic proteins (BMPs) exert important functions in several tissues by regulating signaling for cell differentiation and migration. In vivo, the extracellular matrix (ECM) not only provides support for adherent cells, but also acts as reservoir of GFs. Several constituents of the ECM provide adhesive cues, which serve as binding sites for cell trans-membrane receptors, such as integrins. In conveying adhesion-mediated signaling to the intracellular compartment, integrins do not function alone but rather crosstalk and cooperate with other receptors, such as GF receptors. Here, we present a strategy for the immobilization of BMP-2 onto cellular fibronectin (cFN), a key protein of the ECM, to investigate GF-mediated signaling and migration. Following biotinylation, BMP-2 was linked to biotinylated cFN using NeutrAvidin as cross-linker. Characterization with quartz crystal microbalance with dissipation monitoring and enzyme-linked immunosorbent assay confirmed the efficient immobilization of BMP-2 on cFN over a period of 24 h. To validate the bioactivity of matrix-immobilized BMP-2 (iBMP-2), we investigated short- and long-term responses of C2C12 myoblasts, which are an established in vitro model for BMP-2 signaling, in comparison to soluble BMP-2 (sBMP-2) or in absence of GFs. Similarly to sBMP-2, iBMP-2 triggered Smad 1/5 phosphorylation and translocation of the complex to the nucleus, corresponding to the activation of BMP-mediated Smad-dependent pathway. Additionally, successful suppression of myotube formation was observed after 6 days in sBMP-2 and iBMP-2. We next implemented this approach in the fabrication of cFN micropatterned stripes by soft lithography. These stripes allowed cell-surface interaction only on the patterned cFN, since the surface in between was passivated, thus serving as platform for studies on directed cell migration. During a 10-h observation time, the migratory behavior, especially the cells' net displacement, was increased in presence of BMP-2. As such, this versatile tool retains the bioactivity of GFs and allows the presentation of ECM adhesive cues

Selective modulation of cell response on engineered fractal silicon substrates

A plethora of work has been dedicated to the analysis of cell behavior on substrates with ordered topographical features. However, the natural cell microenvironment is characterized by biomechanical cues organized over multiple scales. Here, randomly rough, self-affinefractal surfaces are generated out of silicon,where roughness Ra and fractal dimension Df are independently controlled. The proliferation rates, the formation of adhesion structures, and the morphology of 3T3 murine fibroblasts are monitored over six different substrates. The proliferation rate is maximized on surfaces with moderate roughness (Ra ~ 40 nm) and large fractal dimension (Df ~ 2.4); whereas adhesion structures are wider and more stable on substrates with higher roughness (Ra ~ 50 nm) and lower fractal dimension (Df ~ 2.2). Higher proliferation occurson substrates exhibiting densely packed and sharp peaks, whereas more regular ridges favor adhesion. These results suggest that randomly roughtopographies can selectively modulate cell behavior

Nanoparticle Tension Probes Patterned at the Nanoscale: Impact of Integrin Clustering on Force Transmission

Herein we aimed to understand how nanoscale clustering of RGD ligands alters the mechano-regulation of their integrin receptors. We combined molecular tension fluorescence microscopy with block copolymer micelle nanolithography to fabricate substrates with arrays of precisely spaced probes that can generate a 10-fold fluorescence response to pN-forces. We found that the mechanism of sensing ligand spacing is force-mediated. This strategy is broadly applicable to investigating receptor clustering and its role in mechanotransduction pathways