July 2011, Issue 2 - Research Spotlight

Andreas Holloschi began a research collaboration with the Bruker team two years ago, which rapidly led to promising results in the field of  neurodegenerative diseases (Download the PDF application notes - AFM and neurodegenerative diseases, Part 1 [AN117] and AFM and neurodegenerative diseases, Part 2: [AN118]). In a first study, he focused his attention on Huntington’s disease, a progressive autosomal dominant disorder that is caused by expansion of the glutamine tract of huntingtin (htt) and leads to psychiatric, motor and cognitive impairments. The mechanism underlying the cause of this disease remains unclear, although multiple pathologic mechanisms have been proposed. As in all polyglutamine disorders, Huntington’s disease is characterized pathologically by the presence of polyglutamine protein containing intracellular aggregates. The Bruker's BioScope® Catalyst™ AFM platform features capabilities that allowed the rapid localizing and imaging of such aggregates in living cells. Those results were correlated to data obtained by fluorescence resonance energy transfer (FRET) techniques that can also be used to investigate protein aggregation (see figure 1).

Fig.1 FRET-based cellular assay for the aggregation of mutant huntingtin. FRET is a process that strongly depends on the distance between the donor and the acceptor fluorophore (typically <10nm), which makes it an ideal tool to study processes like aggregation where proteins get in close proximity (A). FRET microscopy acceptor photobleaching (B) of co-expressed htt exon 1 labeled with CFP and YFP revealed that energy transfer was only detectable in aggregates.

The second study was based on the investigation of the binding between an analog of dopamine attached to an AFM tip, and one of its specific receptors located in the plasma membrane of living cells. Dopamine is a neurotransmitter that regulates key functions in the peripheral (cardiovascular flow, hormone turnover, renal function and gastro-intestinal motility) and central (locomotion, cognition, emotion, food intake and endocrine regulation) nervous system. Any abnormal rate of dopamine in the human body can lead to sever afflictions, such as Parkinson’s disease, schizophrenia, paranoia or Tourette’s syndrome. For treatment of these diseases, the identification of dopaminergic drugs devoid of side effects is one of the biggest challenges in neuronal research and drug discovery. By using functionalized AFM tips in Force Volume mode, and correlating this with fluorescence experiments, it was clearly demonstrated that dopamine is internalized in the cell cytosol after binding to its specific receptor.

More recently, Andreas used Bruker’s BioScope Catalyst™ AFM and MIRO (Microscopy Image Registration Overlay) software to successfully study the effect of two drugs that induce disruption of tubulin and actin cytoskeleton inside living cells (see fig2). This work opens the way to many exiting opportunities in the field of cancer research.

Fig.2 Download Bruker application note AN125. Overlay of AFM and epifluorescence images of living Hela cells. MIRO can be used not only to target location for AFM images but also for single force measurements or Force Volume images, which is a key requirement to study changes in cell response to drug injection.

Hyperosmotic stress induces phosphorylation of cytosolic phospholipase A(2) in HaCaT cells by an epidermal growth factor receptor-mediated process.
Rodríguez I, Kaszkin M, Holloschi A, Kabsch K, Marqués MM, Mao X, Alonso A.
Cell Signal. 2002 Oct;14(10):839-48.

Ionomycin-activated calpain triggers apoptosis. A probable role for Bcl-2 family members.
Gil-Parrado S, Fernández-Montalván A, Assfalg-Machleidt I, Popp O, Bestvater F, Holloschi A, Knoch TA, Auerswald EA, Welsh K, Reed JC, Fritz H, Fuentes-Prior P, Spiess E, Salvesen GS, Machleidt W.
J Biol Chem. 2002 Jul 26;277(30):27217-26. Epub 2002 May 8.