Microinjection mutants

Fig. 3. Actinomorphic effect induced by Clostridium difficile ToxB in UDP-GIc-deficient mutant cells after microinjection of UDP-GIc. Chinese hamster lung mutant cells (Florin, 1991) were treated with ToxB (12ng/ml) for 1 h, after which some cells were microinjected with UDP-GIc (100mM). The microinjected cells developed the characteristic actinomorphic effect (Chang ef al., 1978) within 30 min, whereas non-injected cells (arrows) were unaffected (Chaves-Olarte et al., 1996)...

A EXPERIMENTAL FIGURE 7-19 Oocyte expression assay is useful in comparing the function of normal and mutant forms of a channel protein. A follicular frog oocyte is first treated with collagenase to remove the surrounding follicle cells, leaving a denuded oocyte, which is microinjected with mRNA encoding the channel protein under study. [Adapted from I R Smith, 1988, Trends Neurosci. 250.]... 

The first indication that Ras functions downstream from RTKs in a common signaling pathway came from experiments in which cultured fibroblast cells were induced to proliferate by treatment with a mixture of PDGF and EGF. Microinjection of anti-Ras antibodies into these cells blocked cell proliferation. Conversely, injection of Ras , a constitu-tively active mutant Ras protein that hydrolyzes GTP very inefficiently and thus persists in the active state, caused the cells to proliferate in the absence of the growth factors. These findings are consistent with studies showing that addition of FGF to fibroblasts leads to a rapid increase in the proportion of Ras present in the GTP-bound active form. 

Import of aminoacyl-tRNA into living cells is another approach toward in vivo production of nonnatural mutant proteins. Dougherty and coworkers microinjected [41] or electroporated [44] an aminoacyl-tRNA/mRNA pair into Xenopus oocyte to synthesize fluorescently labeled acetylcholine receptor. The microinjection method is applicable to any type of tRNA and amino acid, but the number of cells that can be treated at one time is very limited. 

The assays described in this chapter are designed to quantify the relative rate with which an NLS green fluorescent protein (NLS-GFP) fusion protein moves in and out of the nucleus, in normal and mutant yeast cells, under a variety of physiological and nonphysiological conditions. The assay strategy circumvents the need to microinject substrates, which is not generally feasible in yeast. Al-... 

To control whether lamin A and lamin A mutants labeled with 5-IAF exhibit properties different from native lamins, we routinely tested their in vitro assembly properties prior to microinjection. Lamin A forms in vitro long paracrystals with periodic repeats of 22-25 nm, whereas a mutant lacking the complete carboxy-terminal tail formed filament bundles (for examples, see Schmidt et al., 1994 Schmidt and Krohne, 1995). For microinjection experiments only batches of 5-IAF lamins were used that were, by electron microscopical inspection, indistinguishable in their in vitro assembly properties from the nonlabeled protein. 

The study of lamin assembly by the microinjection of fluorescently labeled lamins has the advantage that controlled amounts of renatured protein can be injected, and that the manipulated cells can be continuously observed. The formation of heterodimeric complexes between microinjected 5-IAF-labeled lamins and the endogenous lamins can be largely excluded because the fluorescently labeled lamins are mainly in the form of homodimers assembled during dialysis. Comparison of our microinjection data with results obtained by transfection experiments demonstrates that the mutant phenotype can be obscured in transfected cells by overexpression and/or the formation of heterooligomeric complexes of mutant molecules with wild-type lamins of the transfected cells (Schmidt and Krohne, 1995). Therefore we believe that transfection experiments performed with cDNAs coding for mutated nuclear proteins should eventually be controlled by the microinjection of fluorescently labeled protein. 

In a pioneering forward chemical genetic screen (whole-cell mitotic arrest assay detected by fluorescence microscopy), a cell-permeable small molecule, monastrol (Figure 1.9), was identified, as it caused inhibition of the normal mitotic spindle formation but did not affect normal tubulin formation. In subsequent studies, testing the inhibition of the formation of the mutant phenotype led to the identification of the primary molecular target in the signaling cascade, a molecular motor protein, kinesin, Eg5. Monastrol treatment showed a phenotype identical to the blocking of Eg5 function by microinjection of Eg5-specific antibodies (Kapoor et al., 2000). 

COPII association with ER membranes (Stroud et al, 2003). Furthermore, it should be noted that in the case of Sari the GTP-restricted protein has been the microinjected protein of choice not because it is the right mutant protein but because it is easier to isolate than the GDP-restricted mutant in stable, high activity form. As a final buyer beware note, the reader is reminded that small GTPase are in general lipid modified and that these modifications typically are not produced when isolating the recombinant, his-tagged protein from E. coli. The experimenter hopes that the injected cell will do what E. coli did not. 

In sum, direct mutant protein microinjection is an attractive choice for which the major hurdle is isolation of the mutant protein as an active, stable protein. 

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