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The photoreceptor outer segment (OS), a well-defined sensory cilium, provides an important context for the study of intraflagellar transport (IFT). The early phases of OS development involve successive events that are common to virtually all cilia. Additionally, intense protein trafficking occurs through the cilium and relies on IFT to maintain proper cellular morphology and optimize the photosensitive function. In the past decade, progress has been made in the characterization of photoreceptor OS trafficking in murine and amphibian models. Recently, powerful and cost-effective molecular tools and techniques for zebrafish have opened new opportunities to study photoreceptor IFT. Studies using zebrafish take advantage of its rapid embryogenesis to characterize the early events involved in photoreceptor ciliogenesis and OS assembly. In this overview, we describe phenotypes associated with knockdown strategies or genetic mutations of IFT components in zebrafish and detail a general experimental approach that has enabled us to study the function of the two anterograde IFT motors, KIF17 and kinesin II, in zebrafish cone photoreceptors.

Classical biochemistry is founded on several assumptions valid in dilute aqueous solutions that are often extended without question to the interior milieu of intact cells. In the first section of this chapter, we present these assumptions and briefly examine the ways in which the cell interior may depart from the conditions of an ideal solution. In the second section, we summarize experimental evidence regarding the physical properties of the cell cytoplasm and their effect on the diffusion and binding of macromolecules and vesicles. While many details remain to be worked out, it is clear that the aqueous phase of the cytoplasm is crowded rather than dilute, and that the diffusion and partitioning of macromolecules and vesicles in cytoplasm is highly restricted by steric hindrance as well as by unexpected binding interactions. Furthermore, the enzymes of several metabolic pathways are now known to be organized into structural and functional units with specific localizations in the solid phase, and as much as half the cellular protein content may also be in the solid phase.

Publisher Summary This chapter presents the study of spatial and temporal changes in the structure and chemistry of cells. The chapter also presents requirements of multiple spectral parameter imaging. It is evident that most cellular functions involve the temporal and spatial integration of changes in ionic parameters, metabolites, macromolecules, and organelles. Therefore, a method has been developed to correlate the temporal and spatial variations in up to five separate parameters based on the spectral isolation of distinct fluorescent probes. This method allows the integration of results from distinct measurements of parameters such as pH, free calcium concentration, membrane potential, specific fluorescent analogs, and whole organelles. The chapter explains the structural and chemical changes in quiescent Swiss 3T3 cells during the polarized spreading and the movement following activation by creating a wound.

Publisher Summary This chapter describes the preparation of fluorescently labeled dextrans and ficolls. The fact that dextran and Ficoll cannot cross biological membranes makes them ideal as markers and probes of specific subcellular compartments. Fluorescein dextran was first reported as a marker for fluid phase pinocytosis. A red-fluorescent, cyanine derivative of dextran has been used to mark the pinosome compartment in a multiparameter study of living Swiss 3T3 cells. The dibutyl tin dilaurate method is convenient and suitable for studies in which the degree of substitution doesn't need to be controlled accurately and detectability of small amounts is the limiting factor. It is found that when the method is used to prepare tetramethylrhodamine (TRTC-) derivatives, a minor complication results, apparently due to the hydrophobicity of the fluorophore. TRTC-dextran is slightly soluble in ethanol, so that repeated washing in ethanol sometimes results in progressive loss of labeled material. The aminoethylcarboxymethyl method involves conversion of hydroxyl groups to O-carboxy-methyl ethers and subsequent amidation of these groups by ethylene diamine in the presence of a water soluble carbodiimide. It is found that the degree of substitution can be determined with reasonable accuracy by reading the absorbance of the fluorophore for a known concentration of fluorescent polysaccharide.