The role of microtubule movement in bidirectional organelle transport

We study the role of microtubule movement in bidirectional organelle transport in Drosophila S2 cells and show that EGFP-tagged peroxisomes in cells serve as sensitive probes of motor induced, noisy cytoskeletal motions. Multiple peroxisomes move in unison over large time windows and show correlations with microtubule tip positions, indicating rapid microtubule fluctuations in the longitudinal direction. We report the first high-resolution measurement of longitudinal microtubule fluctuations performed by tracing such pairs of co-moving peroxisomes. The resulting picture shows that motor-dependent longitudinal microtubule oscillations contribute significantly to cargo movement along microtubules. Thus, contrary to the conventional view, organelle transport cannot be described solely in terms of cargo movement along stationary microtubule tracks, but instead includes a strong contribution from the movement of the tracks.Comment: 24 pages, 5 figure

High-throughput sequencing of Astrammina rara: Sampling the giant genome of a giant foraminiferan protist

<p>Abstract</p> <p>Background</p> <p>Foraminiferan protists, which are significant players in most marine ecosystems, are also genetic innovators, harboring unique modifications to proteins that make up the basic eukaryotic cell machinery. Despite their ecological and evolutionary importance, foraminiferan genomes are poorly understood due to the extreme sequence divergence of many genes and the difficulty of obtaining pure samples: exogenous DNA from ingested food or ecto/endo symbionts often vastly exceed the amount of "native" DNA, and foraminiferans cannot be cultured axenically. Few foraminiferal genes have been sequenced from genomic material, although partial sequences of coding regions have been determined by EST studies and mass spectroscopy. The lack of genomic data has impeded evolutionary and cell-biology studies and has also hindered our ability to test ecological hypotheses using genetic tools.</p> <p>Results</p> <p>454 sequence analysis was performed on a library derived from whole genome amplification of microdissected nuclei of the Antarctic foraminiferan <it>Astrammina rara</it>. Xenogenomic sequence, which was shown not to be of eukaryotic origin, represented only 12% of the sample. The first foraminiferal examples of important classes of genes, such as tRNA genes, are reported, and we present evidence that sequences of mitochondrial origin have been translocated to the nucleus. The recovery of a 3' UTR and downstream sequence from an actin gene suggests that foraminiferal mRNA processing may have some unusual features. Finally, the presence of a co-purified bacterial genome in the library also permitted the first calculation of the size of a foraminiferal genome by molecular methods, and statistical analysis of sequence from different genomic sources indicates that low-complexity tracts of the genome may be endoreplicated in some stages of the foraminiferal life cycle.</p> <p>Conclusions</p> <p>These data provide the first window into genomic organization and genetic control in these organisms, and also complement and expands upon information about foraminiferal genes based on EST projects. The genomic data obtained are informative for environmental and cell-biological studies, and will also be useful for efforts to understand relationships between foraminiferans and other protists.</p

Laser microsurgery in the GFP era : a cell biologist's perspective

Author Posting. © The Author(s), 2007. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Methods in Cell Biology 82 (2007): 237, 239-266, doi:10.1016/S0091-679X(06)82007-8.Modern biology is based largely on a reductionistic ‘dissection’ approach – most cell biologists try to determine how complex biological systems work by removing their individual parts and studying the effects of this removal on the system. A variety of enzymatic and mechanical methods have been developed to dissect large cell assemblies like tissues and organs. Further, individual proteins can be inactivated or removed within a cell by genetic manipulations (e.g., RNAi or gene knockouts). However, there is a growing demand for tools that allow intracellular manipulations at the level of individual organelles. Laser microsurgery is ideally suited for this purpose and the popularity of this approach is on the rise among cell biologists. In this chapter we review some of the applications for laser microsurgery at the subcellular level, and describe practical requirements for laser microsurgery instrumentation demanded in the field. We also outline a relatively inexpensive but versatile laser microsurgery workstation that is being used in our lab. Our major thesis is that the limitations of the technology are no longer at the level of the laser, microscope or software, but instead only in defining creative questions and in visualizing the target to be destroyed.Our work is sponsored by grants from the NIH (GM59363 to AK and GM40198 to CLR) and HFSP (RGP0064 to AK). Construction of the laser microsurgery workstation was supported in par by Summer Research Fellowship from Nikon/Marine Biological Laboratory (2003 to AK)