In Drosophila, enhancer trap strategies allow rapid access to expression patterns, molecular data, and mutations in trapped genes. However, they do not give any information at the protein level, e.g., about the protein subcellular localization. Using the green fluorescent protein (GFP) as a mobile artificial exon carried by a transposable P-element, we have developed a protein trap system. We screened for individual flies, in which GFP tags fulllength endogenous proteins expressed from their endogenous locus, allowing us to observe their cellular and subcellular distribution. GFP fusions are targeted to virtually any compartment of the cell. In the case of insertions in previously known genes, we observe that the subcellular localization of the fusion protein corresponds to the described distribution of the endogenous protein. The artificial GFP exon does not disturb upstream and downstream splicing events. Many insertions correspond to genes not predicted by the Drosophila Genome Project. Our results show the feasibility of a protein trap in Drosophila. GFP reveals in real time the dynamics of protein's distribution in the whole, live organism and provides useful markers for a number of cellular structures and compartments.

Bovine neurotubulin has been labeled with dichlorotriazinyl-aminofluorescein (DTAF-tubulin) and microinjected into cultured mammalian cells strains PTK1 and BSC. The fibrous, fluorescence patterns that developed in the microinjected cells were almost indistinguishable from the pattern of microtubules seen in the same cells by indirect immunofluorescence. DTAF-tubulin participated in the formation of all visible, microtubule-related structures at all cell cycle stages for at least 48 h after injection. Treatments of injected cells with Nocodazole or Taxol showed that DTAF-tubulin closely mimicked the behavior of endogenous tubulin. The rate at which microtubules incorporated DTAF-tubulin depended on the cellcycle stage of the injected cell. Mitotic microtubules became fluorescent within seconds while interphase microtubules required minutes. Studies using fluorescence redistribution after photobleaching confirmed this apparent difference in tubulin dynamics between mitotic and interphase cells. The temporal patterns of redistribution included a rapid phase (~3 s) that we attribute to diffusion of free DTAF-tubulin and a second, slower phase that seems to represent the exchange of bleached DTAF-tubulin in microtubules with free, unbleached DTAF-tubulin. Mean half times of redistribution were 18-fold shorter in mitotic cells than they were in interphase cells.The organization and apparent function of microtubules in cultured mammalian cells vary markedly as the cell cycle proceeds from interphase to mitosis and back to interphase again. Structural studies with polarization optics, immunofluorescence, and electron microscopy have shown that the mitotic spindle is an active structure in which microtubules exhibit rather rapid changes in arrangement and length (l 1-13, 22, 23, 27, 3 l, 38). The function of mitotic microtubules has been the subject of a great deal of debate for many years (reviewed in references 11 and 27), but it is clear that they are involved in the movement of chromosomes. Interphase cytoskeletal microtubules appear comparatively static with changes in arrangement and length occurring slowly (15). Although their function is also the subject of debate, they may be involved in the specification of cell polarity, shape, and cytoplasmic organization (3,22,28,37,44).To understand the different behaviors of microtubules in mitotic and interphase cells, it may be useful to investigate the dynamic relationship between tubulin and microtubules in vivo. Studying this relationship in detail requires a method for viewing tubulin in both polymer and dimer form in living cells. In principle, microinjection of fluorescently labeled tubulin followed by fluorescence microscopy provides such a method. In practice it is a useful method only if the labeled tubulin retains the functional characteristics of the native protein (17,18,36,40). Recent work supports the belief that the behavior of tubulin labeled with dichlorotriazinyl-aminofluorescein (DTAF) is analogous to the behavior of unlabeled tubulin (15,19,...