Lipoxidation of vimentin and its interplay with zinc

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Bioquímica y Biología Molecular, leída el 19-12-2019La vimentina es una proteína perteneciente a la familia de filamentos intermedios de tipo III que se encuentra comúnmente en fibroblastos, leucocitos, otras células de origen mesenquimatoso y en cristalino. A lo largo de los años, se ha descrito que la vimentina participa en una gran variedad de funciones celulares y tisulares y está involucrada en los mecanismos patogénicos de varias enfermedades [1].En células, la vimentina forma una extensa y dinámica trama de filamentos que se extiende por todo el citoplasma, y sufre alteraciones constantes y relativamente rápidas de su organización y localización en respuesta a modificaciones postraduccionales y a estrés bioquímico y / o mecánico...Vimentin is a type III intermediate filament protein commonly found in fibroblasts, leukocytes, other cells of mesenchymal origin and in the eye lens. Over the years, vimentin has been described to play diverse roles across a great variety of cell and tissue functions and to be involved in the pathogenic mechanisms of several diseases [1].In cells, vimentin builds an extended and actively dynamic network of filaments that spans the cytoplasm, and undergoes constant and relatively rapid alterations of shape and localization in response to post-translational modifications and to biochemical and/or mechanical stress...Fac. de FarmaciaTRUEunpu

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

COMBINED ATOMIC FORCE MICROSCOPY AND LIGHT SHEET MICROSCOPY CHARACTERIZE THE MECHANOBIOLOGY OF PHAGOCYTOSIS

Phagocytosis is a fundamental biological function which allows immune cells, such as macrophages, to internalize and eliminate harmful particles such as pathogens, bacteria, and cell debris. These phagocytic targets vary widely in their size, shape, and stiffness requiring unique and complementary modes of clearance. Macrophage cells are able to exert forces to probe for the target’s mechanical properties and respond with changes in their cellular structures allowing for internalization. This force-driven mechanism of sensing a target’s composition has been shown to be an important factor influencing how phagocytosis proceeds. In order to better understand the forces directing cell decision-making before and during phagocytosis, it is imperative to measure the forces exerted on the target by the cell as well as capture high spatiotemporal resolution of the underlying actin cytoskeleton proving the scaffolding for dynamic cell structure changes. In this dissertation, I measure and observe the mechanical activity of the cell by using an Atomic Force Microscope (AFM) combined with Line Bessel Light sheet microscopy to simultaneously measure the force required to pull a target off the AFM cantilever and image the filamentous action (f-actin) location and intensity over the course of engulfment.Doctor of Philosoph

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin

The role of calcium-permeable AMPA receptors and arc in secreted amyloid precursor protein alpha-mediated plasticity

The orchestrated regulation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype of glutamate receptors by neuronal activity and neuromodulators is critical to the expression of both long-term potentiation (LTP) and memory. In particular, GluA1-containing, Ca2+-permeable AMPAR (CP-AMPAR) comprise a unique role in these processes due to their transient, activity-regulated expression at the synapse. Importantly, many of the mechanisms which govern these processes are negatively affected in neurodegenerative disorders such as Alzheimer’s disease, suggesting that understanding the mode of action of neuromodulatory molecules may reveal much needed novel therapeutic interventions. Secreted amyloid precursor protein-alpha (sAPPα), a metabolite of the parent amyloid precursor protein (APP) has been previously shown to enhance hippocampal LTP and facilitate memory formation. Accordingly, we hypothesised that sAPPα may act via modulation of AMPAR synthesis and cell surface expression. Using primary hippocampal neurons grown in culture, we found that sAPPα (1 nM) differentially regulates the expression of cell surface GluA1-, GluA2-, and GluA3-containing AMPAR. Interestingly, using fluorescent non-canonical amino acid tagging with proximity ligation assay (FUNCAT-PLA), we found that short-term sAPPα treatments (1 nM, 30 min) rapidly enhanced the cell surface expression of newly synthesised extrasynaptic GluA1-, but not GluA2-containing AMPAR, while long-term treatments of sAPPα (1 nM, 120 min) increased levels of pre-existing GluA1/2-containing heteromers at the cell surface, indicating a dynamic regulation of distinct AMPARs following treatment. Moreover, using electrophysiology in area CA1 of acute hippocampal slices, we provide evidence that the expression of CP-AMPAR is important in the induction of sAPPα-enhanced LTP. Using immunocytochemistry and siRNA knockdown, we provide evidence that internalization of CP-AMPARs may be governed, at least in part by sAPPα-driven expression of the activity-regulated cytoskeletal-associated protein (Arc). Further, we show that Arc expression is not induced by the related APP metabolite sAPPβ, but is dependent on synergistic activation of N-Methyl-D-Aspartate and α7-nicotinic acetylcholine receptors, as well as downstream activation of CaMKII, MAPK, and PKG. Together, these findings suggest that application of sAPPα to hippocampal neurons engages a cascade of mechanisms which enhance the synthesis and expression of AMPAR and Arc protein, in the regulation of synaptic strength and the expression of hippocampal LTP. These experiments expand upon our current knowledge underlying mechanisms of synaptic plasticity in hippocampal neurons