Robert E. Palazzo

Warming up at the poles

The third EMBO workshop on Centrosomes and Spindle Pole Bodies, organized by M. Bornens, S.J. Doxsey, M. Knop and J. Raff, was held at the European Molecular Biology Laboratory in Heidelberg, Germany, between 23 and 27 September 2005. ![][1] This series of meetings (Palazzo, 2002; Stearns & Winey, 1997) began as a joint ASCB/EMBO venture in 1997 with a meeting that was a landmark event, not only because a minor field had grown to fill an entire meeting, but also because key presentations defined the course of the field for years to come. The first biochemical mass spectrometric analysis of the budding yeast spindle pole body (SPB) by John Kilmartin and Matthias Mann marked the dawn of a new era in centrosome and SPB studies (Wigge et al , 1998). Similarly, Jeff Salisburys clear demonstration of centrosome amplification in tumour samples, coupled with the work of Bill Brinkley, Stephen Doxsey and others, was part of a resurrection of Theodor Boveris turn‐of‐the‐century hypothesis that centrosome amplification could lead to genetic instability and cancer (Brinkley, 2001). However, all was not harmonious, as elegant work describing spindle assembly in centrosome‐free Xenopus extracts (Heald et al , 1996), coupled with studies showing Drosophila development in the absence of functional centrosomes, sparked a key debate at the 1997 meeting that persists to this day. The root of this controversy lies in the ability of spindles to form and apparently function without centrosomes. Mass spectrometry continued to set the pace at the second meeting in 2002, as Erich Nigg and Manns groups defined a set of centrosome components that have become the staple diet for the community ever since (Andersen et al , 2003). However, more contention arose at the second meeting as a result of the stunning laser ablation studies by Alexy Khodjakov that challenged another of Boveris … [1]: /embed/graphic-1.gif

Laser-Induced Nanopores in Living Cells

We report the creation of nanometer-sized artificial pores in membranes of living cells by femtosecond near-infrared laser pulses. This is a novel approach to deliver molecules into which are resistant to conventional microinjection techniques.

Femtosecond near-infrared opto-injection of single living cells: pore size in dependence of laser intensity

We investigate the size of transient artificial pores as a function of the incident laser intensity in femtosecond nearinfrared laser opto-injection into single living Bovine aortic endothelial cells (BAECs). Molecules ranging in size from 457 dalton to 500kD were used in size exclusion experiments. We found that the threshold laser intensity for pore creation was in dependence of the size of the molecule.

Microinjection by femtosecond near-infrared laser pulses

A Titanium-sapphire laser emitting 100fs short laser pulses at 800nm wavelength is employed to inject an impermeable dye (Lucifer Yellow) into single Bovine Aortic Endothelial cells. The cell morphology remains intact after this process. A new method to analyze dye uptake and evaluate membrane integrity is suggested.