Scientists came from around the world to present at ASCB’s joint meeting with the International Federation for Cell Biologists (IFCB) in Philadelphia December 6-10. This year’s stellar science presentations included fascinating insights into cellular mechanisms, exciting translational approaches, and cutting-edge biophysics. This is Part One of our ASCB/IFCB 2014 roundup.
Cell Biological Approaches to Understanding Disease
A Spotlight on Cancer
If the soles of your shoes say something about where you’re going, the footprint of a cell can give clues about cancer. This idea of tracking something small, like a molecular footprint, to understand something big, like cancer progression, was the theme of one of the 22 special interest subgroup sessions in Philadelphia. The member-organized subgroups kicked off the science at the meeting Saturday afternoon. The “Spanning Scales (Nano to Macro): Understanding Bigger Things in Biology by Looking at the Smaller Things” session was organized by Cathy Galbraith and Jim Galbraith of Oregon Health and Science University. They look at single adhesion receptor molecules and see how the cell is going to move. Their session also featured talks by Gleb Stengel of the Howard Hughes Medical Institute’s Janelia Farm campus, who is looking at whether superresolution imaging can correlate with electron microscopy, and Michael Reiser of HHMI Janelia Farm, who is recording electrical signals and relating that back to animal behavior.
More progress toward elucidating cancer was presented in the ASCB Learning Center the following day. Jae-Won Shin, a postdoc in David Mooney’s lab at Harvard University’s Wyss Institute for Biologically Inspired Engineering in Cambridge, MA, presented a poster on building a three-dimensional (3D) hydrogel system with tunable stiffness to see how relative stiffness of the surrounding ECM affected the resistance of human myeloid leukemias to chemotherapeutic drugs. Shin and Mooney found, for example, that chronic myeloid leukemias (CML) grown in their viscous 3D gel system were more resistant to a widely used cancer drug, Imatinib (Gleevec), than those cultured in a rigid matrix. Using this and other data from their variable ECM system, the researchers screened libraries of small molecule drugs, identifying a subset of drugs they say will be more likely to be effective against CML, regardless of the surrounding matrix. By correcting for the matrix effect, Shin believes this novel approach to drug screening could more precisely tailor chemotherapy to a patient’s individual blood cancer type. They also looked at a cellular signaling pathway, Protein Kinase B (AKT), known to be involved in mechanotransduction and thus sensitive to stiffness in different leukemia subtypes. They discovered that CML cells in the 3D hydrogel were resistant to an AKT inhibitor while AML cells grown in the same conditions were responsive to the drug, supporting their idea that a tunable matrix system could be a way to sort out subtypes by drug resistance.
Before cells become cancerous they must invade the basement membrane, but how? There are carcinoma-associated fibroblasts (CAF) whose matrix proteinases could come in handy in breaking the membrane barrier. The question becomes who is invading whom—do cancer cells invade the basement membrane or do some fibroblasts help invading cancers? Alexandros Glentis of the Institut Curie in Paris presented evidence at the “Mechanotransduction of Disease” minisymposium on December 10 of a coordinated attack on the basement membrane by cancer cells in situ and CAF cells in the extracellular matrix. Using human colon cancer cells and primary human fibroblasts isolated from tumors and adjacent normal tissues, Glentis compared CAFs from colon tumors to normal fibroblasts (NAFs) that were isolated from the same patient, in the adjacent normal tissue. In co-culture experiments on coated transfilters, both NAFs and CAFs induced migration and invasion of HT29, which are intrinsically noninvasive colon cancer cells. Glentis then devised an assay that deployed native basement membrane to separate cancer cells on one side and fibroblasts embedded in collagen on the other. They found that only CAFs were able to stimulate invasion of cancer cells. Applying proteomic analysis confirmed that CAFs secrete more proteases, extracellular matrix proteins, and proteins that modify the basement membrane compared with NAFs, pointing to a remodeling role for CAFs in invasion.
New Disease Models
The search for a living laboratory model of human neurons in the grip of Alzheimer’s disease (AD)—the so-called “Alzheimer’s in a dish”—has a new candidate. Håkan Toresson of Lund University in Sweden reported in his poster on December 9 success in creating induced neurons that model Alzheimer’s by starting with fibroblasts taken from skin biopsies. The differentiated cells express a full range of normal neuronal markers. Significantly, all the neurons derived from fibroblasts including those taken from patients diagnosed with AD, express the proteins classically associated with the neurodegenerative disorder including amyloid beta (Aβ) and the microtubule-associated protein tau, giving researchers a ready comparison between AD patients and the normal elderly. All the fibroblast samples in Toresson’s study came from patients enrolled in a large, long-term clinical research program on dementia called the Swedish BioFinder Study. The biopsies are linked to patient histories and results from batteries of longitudinal tests covering behavior, spinal fluid biochemistry, and brain imaging results including PET and MRI scans. Toresson and colleagues subjected the fibroblast samples to growth factor treatment and transduction by three transcription factors, Ascl1, Brn2 and Myt1l, that induced differentiation into nerve cells. So far Toresson reports success in 12 of 12 attempts to create induced neurons from the BioFinder samples, giving the researchers a cell biological library of neurons from elderly Swedes who exhibit signs of sporadic AD, familial AD, or robust cognitive health. Toresson hopes to compare the cell phenotypes on induced neurons derived from sporadic AD patients with induced neurons from familial AD cases that have the characteristic APP, PSEN1, or PSEN2 mutations. Such an approach could pave the way for personalized treatments for dementia, allowing the use of a patient’s accessible skin cells to assess what’s happening inside the inaccessible neurons of the brain.
The human blood-brain barrier (BBB) separates circulating blood from the central nervous system, thus protecting the brain from many infections and toxins. But the BBB also blocks the passage of many potentially useful drugs to the brain and it has long stymied scientists who want to learn more about this vital tissue because of the lack of realistic non-human lab models. Even less is known about the BBB in children. Postdoctoral fellow Sudhir Deosarkar in the laboratory of Mohammad Kiani at Temple University in Philadelphia presented his experimental workaround—a synthetic pediatric blood-brain barrier on a small chip—in a poster on December 8. He has tested it successfully using rat brain endothelial cells (RBECs) from rat pups and human endothelial cells. Deosarkar calls his physiologically realistic in vitro pediatric BBB model on a chip, the B3C. It has two compartments, one to grow blood vessel cells in and another for cultured brain cells, mimicking the physiology of the BBB. Deosarkar and colleagues fabricated the B3C using an optically clear, oxygen permeable polymer, polydimethylsiloxane, on a glass slide with vascular (apical) and tissue (basolateral) compartments. By culturing RBECs and human endothelial cells under flow conditions, Deosarkar found that cell-cell junctions they formed accurately mimicked endothelial barrier formation in the brain.
Membrane Trafficking in Disease
Philadelphia was an apt location to give a talk about Legionella pneumophila. The Legionella bacterium was first discovered in 1977 after a deadly outbreak of a mystery illness at a Philadelphia meeting of the American Legion at the Bellevue-Stratford Hotel in 1976. Two hundred twenty one of the convention attendees developed flu-like symptoms and 34 later died of pneumonia. The illness, hence named Legionnaires’ disease, was transmitted via the air conditioning system of the hotel. Ventilation standards have been tightened in the U.S. but Legionnaires’ disease is still common in developed countries, yet how Legionella evades the host’s defenses is still not precisely understood. Matthias Machner, investigator at the NIH Eunice Kennedy Shriver National Institute for Child Health and Human Development, shared a piece of the answer in the “Reduce, Reuse, Recycle-The Many Strategies of Microbial Pathogens” minisymposium. Machner described how Legionella uses an effector protein called VipD to disable the endo-lysosomal pathway by first locating a critical protein, Rab5, and then by snipping a nearby lipid that serves as a signpost for endosome formation.
AD progresses inside the brain in a rising storm of cellular chaos as deposits of the toxic protein, amyloid-beta (Aβ), overwhelm neurons. An apparent side effect of accumulating Aβ in neurons is the fragmentation of the Golgi apparatus, the part of the cell involved in packaging and sorting protein cargo, including the precursor of Aβ. But is the destruction the Golgi a kind of collateral damage from the Aβ storm or is the loss of Golgi function itself part of the driving force behind Alzheimer’s? Gunjan Joshi, a postdoctoral fellow in Yanzhuang Wang’s lab at the University of Michigan, Ann Arbor, set out to uncover the mechanism damaging the Golgi, using a transgenic mouse and tissue culture models of AD. He presented his data in a poster session showing that rising levels of Aβ do lead directly to Golgi fragmentation by activating a cell cycle kinase, cdk5. The surprising part of the answer was that Golgi function can be rescued by blocking cdk5 or shielding its downstream target protein in the Golgi, GRASP65. The even more surprising answer was that rescuing the Golgi reduced Aβ accumulation significantly, apparently by re-opening a normal protein degradation pathway for the amyloid precursor protein (APP). To Joshi this suggested an entirely new line of attack for drugs hoping to slow AD progression. He says that Golgi fragmentation is in itself a major—and until now an unrecognized—mechanism through which Aβ extends its toxic effects. Joshi and colleagues believe that as Aβ accumulation rises, damage to the Golgi increases, which in turn accelerates APP trafficking, which in turn increases Aβ production.
Next from ASCB/IFCB 2014—New Biophysical Approaches to Cell Biology Plus Classic Cell Biology at Its Best