SR-SIM, FRET, FRAP
Teaching, Learning and Community
The Neuro Research Group (NRG), headed up by Dr Ben Loos, combines cell biology, cell physiology, microscopy and biochemistry approaches to dissect and investigate the relationship and role of protein degradation through macroautophagy and cell death susceptibility in neurodegeneration, neuronal migration and gliomas. The fine balance of proteostasis control, protein aggregation and proteotoxicity is here of major interest, due to its role in disease onset and progression. Autophagy is an essential process that allows cellular survival in stress conditions through the degradation of long-lived proteins. Since the vast majority of proteins in the cell are long-lived, its metabolic contribution is significant. Dysfunction in autophagy is associated with numerous pathologies that are characterized by a shift in cell death susceptibility, such as neurodegenerative diseases or cancer.
In order to achieve this, the lab focuses on macroautophagy (MA), chaperone mediated autophagy (CMA), cellular metabolism, mitochondrial morphology and function, tubulin and transport systems, the cytoskeleton and ATP consumption. Central to our approach is a dynamic perspective on the cell’s function and its stress response, in context of its current intracellular and extracellular metabolic parameters (Figure 1). Our research findings in the past have demonstrated a clear relationship between the cell’s autophagic proficiency and apoptosis/necrosis onset, which demands for a dynamic and integrative approach.
Many questions in this context remain to be answered. We are interested in the factors that drive cell death susceptibility in Alzheimer’s disease, in order to exploit mechanisms for neuronal protection. We know that a number of pathways related to autophagic flux, protein degradation, axonal transport, mitochondrial fusion & respiration as well as tubulin stability are implicated. Central again is ATP availability in the compartmentalized regions where the ATP demand is highest (Figure 2). Pathways for neuronal migration and plasticity rely on a highly functional actin, tubulin, mitochondrial network interplay, which allows us to better understand the susceptibility for undergoing a network collapse and metabolic failure, changing the cell’s matrices for apoptosis or necrosis onset. Lastly, we aim to exploit the relationship between ATP availability and apoptosis onset in context of astrocytomas, in order to maximize cell death through sensitization. Central to all of the above is the accurate and robust quantification of autophagic flux, the rate of protein degradation through autophagy, in order to control it with highest degree of precision.
These studies will provide important information about cell death and targeting autophagy in cellular physiology.
Research direction and focus
1. Role of autophagy in pathogenesis:
1a. Autophagic flux in context of neurotoxicity
1b. Autophagic activity in substrate metabolism mitochondrial function
1c. Chloroquine, nutrient starvation and ketone bodies on autophagic flux
1d. Autophagy in glioma metabolism and cell death susceptibility
2. Regulation and control of autophagic flux
2a. Autophagic flux control
2b. Autophagic flux and cell death prediction
2c. Method development for measuring autophagic flux
2d. Method development for employing super-resolution and correlative microscopy to assess autophagy
Hons: Akile Khoza (firstname.lastname@example.org)
Hons: Khaalid Khan (email@example.com)
Hons: Danielle Millar (firstname.lastname@example.org)
MSc: Jurgen Kriel (email@example.com)
MSc: Yigael Powrie (firstname.lastname@example.org)
PhD: Andre Du Toit (email@example.com)
PhD: Dumisile Lumkwana (firstname.lastname@example.org)
PhD: Claudia Matlakala Ntsapi (email@example.com)
PhD: Phunya Bhat (firstname.lastname@example.org)
Microscopy Method development/PhD: Lize Engelbrecht (email@example.com)
Post-Doctoral Fellow: Dr Chrisna Swart (firstname.lastname@example.org)
We have learnt that many crucial cell physiological parameters associated with cell death susceptibility are currently often primarily described based on morphology, with less focus on extracting numerical data based on that very morphology. Hence, we approach our research questions, where suitable, with an additional interdisciplinary angle with expertise in biophysics, theoretical physics, mathematical modeling and systems biology, to fill the gaps that classical cell physiology is less able to address. We often rely heavily on microscopy techniques, and utilize cutting edge techniques such as life cell imaging, fluorescence resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP) or super-resolution structured illumination microscopy (SR-SIM) to generate data that can be utilized for statistical analysis (Figure 3 and 4). SIM, which allows us to achieve a resolution of 80nm, is thereby uniquely positioned to generate data that cannot be resolved through confocal imaging. By exploiting the functional aspects of fluorochromes, their spectral sensitivity in a defined environment (pH, voltage across membranes etc) we are able to tighten the gap between light microscopy and electron microscopy.
These techniques are aimed to assist us in describing spatiotemporal detail on a molecular scale, such as protein-protein interactions, structural organization of organelles or localization of key molecules, required to generate dynamic or novel data. Important for us is here to relate these data to cellular function/dysfunction, as this is the heart of physiological sciences.
This is an extremely exciting and new field that we are currently developing in collaboration with Prof Müller-Nedebock and Dr Leandro Boonzaaier, so that aspects can tailor-fit into unresolved questions of the above core focus areas. The major strength of such nano-biophysics (http://www.physics.sun.ac.za/~kkmn/nanobio/) approach lies in its capabilities to draw onto numerical data within given constraints, to generate and calculate statistics on patterns and behaviors. If linked to cellular or organelle function and tested on real experimental data, powerful predictions can be established. We are here particularly interested in the emerging field of organelle network analysis related to properties such as elasticity, connectivity and efficiency that report on molecular interactions and cellular function. Current projects address fusion dynamics between autophagosomes and lysosomes, mitochondrial network connectivity and aktin-cyctoskeletal stiffness utilizing superresolution structured illumination imaging and analysis (Figure 5).
We have learnt in the last few years that the “magic bullet” approach for the treatment of complex diseases such as AD or cancer has most often failed. One of the argued reasons is the revealed interplay and control of the genome read-out as a function of inscriptions that are based on an active physiological process (histone code, methylation, metabolic and environmental epigenetics). The reductionist causal chain from genotype to phenotype is currently increasingly completed by numerous downward forms of causation. This demands a shift in our research approach. Systems biology is here uniquely equipped to address questions central to cellular physiology, such as structural complexity, emergent properties and complex network regulatory mechanisms. Our group includes therefore a systems biology approach, where suitable, to unravel the complex control of function. In particular, we currently focus here on the flux assessment and control analysis of protein degradation through autophagy. In order to exploit the autophagic machinery in the clinical setting, reproducible models with modular approaches based upon data and modeling standards are required. In collaboration with the Centre for Studies in Complexity, headed up by Prof Jannie Hofmeyr (http://stbweb02.stb.sun.ac.za/complexity/), we are developing tools to understand the control of autophagic flux (as opposed to its regulation), under utilization of powerful mathematical in silico modeling approaches.
A Neethling, J Mouton, B Loos, V Corfield, C de Villiers and C Kinnear, Filamin C: A novel component of the KCNE2 interactome during hypoxia. Cardiovascular Journal of Africa, 27 (1); 2016: 4-11.
Celia van der Merwe, B Loos and S Bardien. Curcumin rescues a PINK1 knock down SH-SY5Y cellular model of Parkinson's disease from mitochondrial dysfunction and cell death. Molecular Neurobiology. In press.
G van Niekerk, B Loos, T Nell and AM Engelbrecht. Cancer tolerance, resistance, pathogenicity and virulence – deconstructing the disease state. Future Oncology, in press.
W Haylett, C Swart, F H. Van Der Westhuizen, H Van Dyk, L van der Merwe, C van der Merwe, B Loos, J Carr, C Kinnear and S Bardien. Altered mitochondrial respiration and other features of mitochondrial function in parkin-mutant fibroblasts from Parkinson’s disease patients. Parkinson's Disease. 2016. In press
G van Niekerk, B Loos, T Nell and AM Engelbrecht. Autophagy - A free meal in sickness associated anorexia. Autophagy. 2016. In press.
Klionsky, Daniel J., et al. "Guidelines for the use and interpretation of assays for monitoring autophagy." Autophagy 12.1 (2016): 1-222.
Leisching, Gina, et al. "Bcl-2 confers survival in cisplatin treated cervical cancer cells: circumventing cisplatin dose-dependent toxicity and resistance." Journal of Translational Medicine 13.1 (2015): 328.
J Govender, B Loos and A-M Engelbrecht. Melatonin: a protective role against doxorubicin-induced cardiotoxicity Future Oncology 07/2015; 11(14):2003-6. DOI:10.2217/fon.15.48
G Leisching, B Loos, M H Botha and A-M Engelbrecht. The Role of mTOR During Cisplatin Treatment in an in vitro and ex vivo Model of Cervical CancerToxicology (Impact Factor: 3.75). 07/2015; 335. DOI: 10.1016/j.tox.2015.07.010
G Leisching, B Loos, T Nell and A-M Engelbrecht. Sutherlandia frutescens treatment induces apoptosis and modulates the PI3-kinase pathway in colon cells. South African Journal of Botany 04/2015; 100:20-26.
Zahra Zakeri, Ben Loos and Richard A Lockshin. Cell Death during Developmental Processes. eLS. Vol 10; 2015. John Wiley & Sons, Ltd: Chichester.
Jenelle Govender , Ben Loos & Anna-Mart Engelbrecht. Melatonin: a protective role against doxorubicin-induced cardiotoxicity. Future Oncology 2015; 11(14): 2003–2006.
Leon M. T. Dicks, Marlie Botha, Ben Loos and Carine Smith. Adhesion of Lactobacillus reuteri strain Lr1 to equine epithelial cells and competitive exclusion of Clostridium difficile from the gastro-intestinal tract of horses. Annuals of Microbiology. 2015; 65: 1087-1096.
C van der Merwe, Z Jalali Sefid Dashti, A Christoffels, B Loos, and S Bardien. Evidence for a common biological pathway linking three Parkinson's disease-causing genes: parkin, PINK1 and DJ-1. European Journal of Neuroscience 2015. doi: 10.1111/ejn.12872.
G Leisching, B Loos, M Botha and A-M Engelbrecht. A Nontoxic Concentration of Cisplatin Induces Autophagy in Cervical Cancer: Selective Cancer Cell Death With Autophagy Inhibition as an Adjuvant Treatment. International Journal of Gynecological Cancer 2015. 25(3):380-388.
J Mouton, B Loos, J C Moolman-Smook, C J Kinnear. Ascribing novel functions to the sarcomeric protein, myosin binding protein H (MyBPH) in cardiac sarcomere contraction, Experimental Cell Research, Volume 331, Issue 2, 15 February 2015, Pages 338-351, ISSN 0014-4827, http://dx.doi.org/10.1016/j.yexcr.2014.11.006·
Loos B. Autophagic Flux and Cell Death. 20 Years of Cell Death. The International Cell Death Society. Editors: Richard A Lockshin and Zahra Zakeri. ISBN: 978-0-9894674-5-2. 2015.
B Loos. Autophagy researchers. Autophagy 2014; 10:1149-1152, PMID: 24905096
B Loos, A Du Toit and JH Hofmeyr. Defining and measuring autophagosome flux-concept and reality. Autophagy 2014, 10:11, 2087-2096, DOI: 10.4161/15548627.2014.973338
J Govender, B Loos, E Marais and A-M Engelbrecht. Mitochondrial catastrophe during doxorubicin-induced cardiotoxicity: a review of the protective role of melatonin. Journal of Pineal Research 2014, 57: 367–380. doi: 10.1111/jpi.12176
T A Davis, B Loos, A-M Engelbrecht, AHNAK: The giant jack of all trades, Cellular Signalling, Volume 26, Issue 12, December 2014, Pages 2683-2693, ISSN 0898-6568, http://dx.doi.org/10.1016/j.cellsig.2014.08.017
C Swart, W Haylett, C Kinnear, G Johnson, S Bardien and B Loos, Neurodegenerative disorders: Dysregulation of a carefully maintained balance?, Experimental Gerontology, Volume 58, October 2014, Pages 279-291, ISSN 0531-5565, http://dx.doi.org/10.1016/j.exger.2014.09.003
B Kleemann, B Loos, T Scriba, D Lang and L Davids. St John's Wort Photomedicine: Hypericin-Photodynamic Therapy induces Metastatic Melanoma Cell Death. PLOS ONE, 2014 (IF: 3.7)
K Jovanovic, B Loos, B Da Costa, C Penny and S Weiss. High resolution imaging study of interactions between the 37kDA/67kDa Laminin receptor and APP, beta-secretase and gamma-secretase in Alzheimer’s disease. PLOS ONE, in press
C van der Merwe, B Loos, C Swart, C Kinnear, F Henning, L van der Merwe, K Pillay, N Muller, D Zaharie, L Engelbrecht, J Carr, S Bardien. Mitochondrial impairment observed in fibroblasts from South African Parkinson's disease patients with parkin mutations. Biochemical and biophysical research communications, 2014.
Loos B, Hofmeyr JH, Müller-Nedebock K, Boonzaaier L and Kinnear C. Autophagic flux, fusion dynamics and cell death. Autophagy: Cancer, Other Pathologies, Inflammation, Immunity, Infection, and Aging, 1st Edition, Volume 3 - Mitophagy. Edited by E. Hayat. ISBN 9780124055292. Elsevier Inc., 2014.
Loos B, Sishi B and Engelbrecht AM. Role of autophagy in heart disease. Autophagy. Autophagy: Cancer, Other Pathologies, Inflammation, Immunity, Infection, and Aging. Volume 2: Role In General Diseases. Edited by E. Hayat. ISBN: 978-0-12-405877-4, Elsevier Inc., 2014.
Loos B, Lockshin R, Klionsky D, Engelbrecht A-M and Zakheri Z. On the variability of autophagy and cell death susceptibility. Autophagy. 2013; 10.
Loos B, Sishi B and Engelbrecht AM. Role of autophagy in heart disease. Autophagy. E. Hayat. Springer Verlag. 2013
Sishi B, Loos B, van Rooyen J and Engelbrecht AM. Doxorubicin induces protein ubiquitination and inhibits proteasome activityduring cardiotoxicity. Toxicology; 2013; 309: 23– 29.
“Have fun” and “Go for it” are very common phrases in our group attitude. It is important to be excited about the work conducted and in doing so to generate creative perspectives. At the same time, it is equally crucial to provide a serious support structure and environment in which skills training, scientific writing and presentation skills but also critical and independent thinking skills can grow and flourish, allowing not only a successful graduation in an appropriate time, at a globally highly competitive level, but also growth as individual person.
Two statements that have been made many years ago strongly resonate with my research philosophy:
“Measure what is measurable, and what is not measurable, make measurable” (Gallileo Gallilei, Italian Physicist, Philosopher, Astronomer, 1564-1642).
Many aspects of our work are currently waiting to be made measurable. The recent advances in techniques, hard ware, probes and computational power needs to be harnessed for that purpose, allowing us to derive unique data that are of relevance to our research focus.
“This application of mathematics to natural phenomena is the aim of all science, because the expression of the laws of phenomena should always be mathematical.” (Claude Bernard, french Physiologist, 1813-1878).
This famous physiologist, who described the concept of homeostasis, already indicated the need to express data in a mathematical manner; however, much information was simply not yet available at his time. Today we have the technologies that could provide many more of the required data points, but we often run the risk of “drowning in the sea of data” (genomics, metabolomics, proteomics), potentially losing sight of the larger picture. This above statement forces to focus on what is really required to answer questions relevant to our research focus.
Please contact Ben Loos at email@example.com for information regarding the availability of Hons, MSc and PhD positions.
Centre for Studies in Complexity (http://stbweb02.stb.sun.ac.za/complexity)
Central Analytical Facility (www.sun.ac.za/saf)
Ben Loos, firstname.lastname@example.org
Department of Physiological Sciences
Merriman Avenue, Mike de Vries Building
Tel: +27 21 808 9196
Fax: +27 21 808 3145
As former unit manager of the Cell Imaging Unit, Central Analytical Facility (CAF, www.sun.ac.za/saf), our group is continuously supporting and developing techniques that could be of potential benefit to many other user groups. Having installed the first SR-SIM superresolution platform in South Africa, it is important to us to foster skills development in these techniques. Training initiatives (http://blogs.sun.ac.za/news/2012/10/16/cutting-edge-microscope-opens-up-new-research-possibilities/) associated with CAF are therefore regularly conducted on a regional and national scale, addressing national research focus areas such as water analysis, TB and malaria research, specific techniques (FRET and FRAP) or generic imaging techniques.
Some techniques that our lab wishes to develop in the near future are:
Correlative light and electron microscopy using SR-SIM, PALM and STORM
FRET through Acceptor bleaching and pFRET calculation
Fluorescence/Raster Image Correlation Spectroscopy (FCS) and RICS
Teaching, Learning, Community, Collaborations
Communication of research findings and activities to scientific peers and also the non-science community is important. Disseminating our research findings through participation in local, national and international conferences and symposia is therefore integral part. Multiple national and international collaborations have been initiated.
Stellenbosch University, SA
University of Pretoria, SA
Queens College, NY, US
Kyoto University, Japan
Nanyang Technological University, Singapore
Sri Jayachamarajendra College of Engineering, India
University of Ibadan, Nigeria
We often engage with the community around us through initiatives related to career guidance for learners, outreach programmes or training initiatives.
Teaching, Learning, Community, Collaborations
Collaborative International workshop: ‘Fluorescence Microscopy – Acquisition, Processing and Analysis’, at Mysore University, Karnataka, India, February 2016.
Neuro Research Group 2015
NRG MICROGRAPH OF THE MONTH: April 2016
Mitophagy in MEF cells – a rare event under control conditions.
Yigael Powrie wins at the MSSA 2014 the ‘Most upcoming microscopist’ award for
‘INVESTIGATING TAU PATHOLOGY IN AN IN VITRO MODEL FOR ALZHEIMER'S DISEASE’.
Jurgen Kriel wins the poster prize at the International Mitochondrial Physiology Conference, March 2015, Cape Town for
‘QUANTIFICATION OF MITOCHONDRIAL FISSION AND FUSION THROUGH PHOTOACTIVATION IN GLIOBLASTOMA MUTLIFORME’.
Jurgen demonstrating photo-activation during the fluorescence microscopy workshop at Mysore University, India, February 2016.