Over milieu & zekerheid

Deel 1 "Natuur en milieu" in studieboek voor middelbaar toeristisch en recreatief onderwijs. Deel 1 dekt alle mbo-kwalificaties duurzaam toerisme

Optogenetic control of organelle transport and positioning

Proper positioning of organelles by cytoskeleton-based motor proteins underlies cellular events such as signalling, polarization and growth. For many organelles, however, the precise connection between position and function has remained unclear, because strategies to control intracellular organelle positioning with spatiotemporal precision are lacking. Here we establish optical control of intracellular transport by using light-sensitive heterodimerization to recruit specific cytoskeletal motor proteins (kinesin, dynein or myosin) to selected cargoes. We demonstrate that the motility of peroxisomes, recycling endosomes and mitochondria can be locally and repeatedly induced or stopped, allowing rapid organelle repositioning. We applied this approach in primary rat hippocampal neurons to test how local positioning of recycling endosomes contributes to axon outgrowth and found that dynein-driven removal of endosomes from axonal growth cones reversibly suppressed axon growth, whereas kinesin-driven endosome enrichment enhanced growth. Our strategy for optogenetic control of organelle positioning will be widely applicable to explore site-specific organelle functions in different model systems

Cell death related genes induced by IR in <i>yw</i> (≥1.5-fold, p<0.005).

<p>The values shown are log₂. The cut-off values were 1.5 fold or more (log₂ of 0.585 or greater) with p<0.005 compared to un-irradiated controls, at 2 hr or 18 hr after irradiation or both. ‘–’ = the gene was not significantly induced with respect to neither p-value or fold change. p-value of 0 means p<1e-10. y = <i>yw</i> control; p = <i>p53</i> mutants, ‘−’ = −IR (0 R); ‘+’ = +IR (4000 R); 2 = 2 hr after irradiation, 18 = 18 hr after irradiation. If there is data for more than one probe set is available for a gene, the set with the best p value was considered.</p

Functional Annotation Clustering of genes induced 2-fold or greater by IR in wild type (<i>yw</i>) wing imaginal discs (p<0.001).

<p>Only clusters with Enrichment Score of >1.3 are shown. Gene ontology information is from DAVID Bioinformatics Resources 6.7, NIAID/NIH (ttp://david.abcc.ncifcrf.gov/). Clusters present in both 2 hr and 18 hr time-points are in bold font.</p

Validation by quantitative RT-PCR of 10 candidate genes identified in microarray analysis.

<p>Total RNA was isolated from wing imaginal discs of third instar larvae at 2 or 18 hr after exposure to 0 or 4000R of X-rays. RNA was reverse-transcribed into cDNA and subjected for quantitative RT-PCR analysis as described in Methods. a-tubulin levels were used to normalize the graphs. Error bar = 1 STD. (A) A schematic representation of data comparisons used to select candidates for validation. The gray box denotes the time at which apoptosis becomes detectable in <i>p53</i> mutants. (B) Q-RT-PCR results in <i>p53</i> mutant wing imaginal discs (B) Q-RT-PCR results in wild type (<i>yw</i>) wing imaginal discs.</p

Light-controlled intracellular transport in Caenorhabditis elegans

To establish and maintain their complex morphology and function, neurons and other polarized cells exploit cytoskeletal motor proteins to distribute cargoes to specific compartments. Recent studies in cultured cells have used inducible motor protein recruitment to explore how different motors contribute to polarized transport and to control the subcellular positioning of organelles. Such approaches also seem promising avenues for studying motor activity and organelle positioning within more complex cellular assemblies, but their applicability to multicellular in vivo systems has so far remained unexplored. Here, we report the development of an optogenetic organelle transport strategy in the in vivo model system Caenorhabditis elegans. We demonstrate that movement and pausing of various organelles can be achieved by recruiting the proper cytoskeletal motor protein with light. In neurons, we find that kinesin and dynein exclusively target the axon and dendrite, respectively, revealing the basic principles for polarized transport. In vivo control of motor attachment and organelle distributions will be widely useful in exploring the mechanisms that govern the dynamic morphogenesis of cells and tissues, within the context of a developing animal

Myosin-V Induces Cargo Immobilization and Clustering at the Axon Initial Segment

The selective transport of different cargoes into axons and dendrites underlies the polarized organization of the neuron. Although it has become clear that the combined activity of different motors determines the destination and selectivity of transport, little is known about the mechanistic details of motor cooperation. For example, the exact role of myosin-V in opposing microtubule-based axon entries has remained unclear. Here we use two orthogonal chemically-induced heterodimerization systems to independently recruit different motors to cargoes. We find that recruiting myosin-V to kinesin-propelled cargoes at approximately equal numbers is sufficient to stall motility. Kinesin-driven cargoes entering the axon were arrested in the axon initial segment (AIS) upon myosin-V recruitment and accumulated in distinct actin-rich hotspots. Importantly, unlike proposed previously, myosin-V did not return these cargoes to the cell body, suggesting that additional mechanism are required to establish cargo retrieval from the AIS