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

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

26th Annual Computational Neuroscience Meeting (CNS*2017): Part 3 - Meeting Abstracts - Antwerp, Belgium. 15–20 July 2017

This work was produced as part of the activities of FAPESP Research,\ud Disseminations and Innovation Center for Neuromathematics (grant\ud 2013/07699-0, S. Paulo Research Foundation). NLK is supported by a\ud FAPESP postdoctoral fellowship (grant 2016/03855-5). ACR is partially\ud supported by a CNPq fellowship (grant 306251/2014-0)

Multiscale Simulations of Biological Membranes : The Challenge To Understand Biological Phenomena in a Living Substance

Biological membranes are tricky to investigate. They are complex in terms of molecular composition and structure, functional over a wide range of time scales, and characterized by nonequilibrium conditions. Because of all of these features, simulations are a great technique to study biomembrane behavior. A significant part of the functional processes in biological membranes takes place at the molecular level; thus computer simulations are the method of choice to explore how their properties emerge from specific molecular features and how the interplay among the numerous molecules gives rise to function over spatial and time scales larger than the molecular ones. In this review, we focus on this broad theme. We discuss the current state-of-the-art of biomembrane simulations that, until now, have largely focused on a rather narrow picture of the complexity of the membranes. Given this, we also discuss the challenges that we should unravel in the foreseeable future. Numerous features such as the actin-cytoskeleton network, the glycocalyx network, and nonequilibrium transport under ATP-driven conditions have so far received very little attention; however, the potential of simulations to solve them would be exceptionally high. A major milestone for this research would be that one day we could say that computer simulations genuinely research biological membranes, not just lipid bilayers.Peer reviewe

Cancer hyperthermia using gold and magnetic nanoparticles

An estimated 12 million people worldwide are diagnosed with cancer every year, with around 17 million cancer-related deaths per year predicted by 2030 (Thun et al. 2010). Contemporary clinical treatments include surgery, chemotherapy and radiotherapy, however all vary in success and exhibit unpleasant side effects. Localised tumour hyperthermia is a moderately new cancer treatment envisaged by researchers, which exploits exclusive tumour vulnerabilities to specific temperature profiles (42-45°C) leading to cancer cell apoptosis, whilst normal tissue cells are relatively unaffected. Hyperthermia is therefore proposed as an alternative potential therapy for cancer, by delivering localised treatment to cancer cells, without the severe side effects associated with traditional therapies. This project aimed to investigate potential hyperthermic treatment of cancer cells in vitro by adopting nanomedicine principles. Inorganic nanoparticles, such as gold or iron oxide, are both capable of generating heat when appropriately stimulated, therefore both have been suggested as candidates for inducing localised tumour heating following their internalisation into cells. In this project, both gold (GNPs) and magnetic (mNPs) were individually assessed for their potential to deliver toxic thermal energy to bone cancer cells (MG63) and breast cancer cells (MCF-7). Studies were carried out both in standard 2D monolayer and in 3D tumour spheroids. When considering use in vivo, it is essential that both GNPs and mNPs are biocompatible, therefore initial studies characterised the cell viability and metabolic activity following incubation with the NPs. The NP internalisation was subsequently verified, prior to hyperthermic studies. Following hyperthermic treatment, both GNPs and mNPs were confirmed as inducing cancer cell death. Further studies were carried out using the GNPs, to identify the cell death pathways activated, where mitochondrial stress was evident following 2D culture tests. Gene and protein expression analysis indicated that cell death occurred predominantly via several apoptotic pathways, through increased fold expression changes in apoptotic markers. Interestingly, cell protective mechanisms were simultaneously switched on, as cells were also observed to exhibit thermotolerance with a number of heat shock proteins (Hsps) being substantially increased during hyperthermic treatments