Antibodies microinjection

Sinard, J.H., Pollard, T.D. (1989). Microinjection into Acanthamoeba castellanii of monoclonal antibodies to mysoin II slows but does not stop cell locomotion. Cell Mot. Cytoskel. 12,42-52. 

That motile Shigella concentrate an ActA homologue on their surface provided a way to identify a proteolytic vinculin fragment as the homologue. Laine et al used synthetic FEFPPPPTDE as an immunogen to generate a polyclonal antibody (Ab). Microinjection of this antibody blocked Shigella motility in infected cells, and the Ab was localized to the bacterial tails. Western blots of... 

Mangeat, P. H., and Burridge, K. (1984). Immunoprecipitation of nonerythrocyte spectrin within live cells following microinjection of specific antibodies Relation to cytoskeletal structures./ Cell. Biol. 98, 1363-1377. 

Mad2 by the microinjection of inhibitory antibodies overrides the checkpoint so that cells enter anaphase in the presence of monastrol with monopolar spindles (25). This finding indicates that the principle of feedback control generally applies to spindle perturbations through highly conserved mechanisms. 

Figure 6 Assay to examine cytokinesis in the presence of a monopolar spindle (25). Treatment with monastrol, a small-molecule inhibitor of the kinesin Eg5, causes cells to arrest in mitosis with monopolar spindles because of activation of the spindle checkpoint. Microinjection of an antibody against the protein Mad2 inactivates the checkpoint so that cells divide with monopolar spindles.

Immunofluorescence techniques are often used to identify specific cellular targets, including proteins, involved in endocytosis. However, because antibody reagents are not ceU permeable, cells typically must be fixed or microinjected to allow binding... 

Fig. 4. Disruption of the synapsin-dependent vesicle pool by presynaptic microinjection of synapsin antibodies in the lamprey reticulospinal synapse. (A) Electron micrograph of a control synapse. (B) A synapse in an axon injected with synapsin antibodies. The axon was lightly stimulated (1 Hz for 12 min) and allowed to rest for 90 min before fixation. Note that a narrow rim of vesicles remains in the antihody-injected synapse. (O Immunogold staining of a reticulospinal synapse with synapsin antibodies. Note that the vesicles adjacent to the presynaptic membrane are almost devoid of gold particles. (D) Visualization of the filamentous cytomatrix that overlaps with the synaptic vesicle pool that remains after perturbation of synapsins. The electron micrograph shows a synapse in a normal axon (i.e. no microinjection or stimulation had been performed) stained with phosphotungstic acid (Gustafsson et al., 1996). The filamentous eytomatrix (arrowheads) at the presynaptic membrane is visible, but not the synaptic vesicle cluster. Designations as in Fig. 1. Scale bar, 0.2 p.m. Reprinted from Brodin et al. (1995) Eur J Neurosci 9 2503-2511, with permission.

Fig. 5. Impairment of high-frequency synaptic transmission after microinjection of synapsin antibodies. (A) Control responses recorded before microinjection of synapsin antibodies at 18 Hz. The plot shows amplitudes of EPSPs evoked in a spinal neuron from a lamprey reticulospinal axon. The traces show averages of EPSPs during the two recording periods (1 and 2, respectively) (B) Responses recorded after microinjection of synapsin antibodies. Note the enhanced depression after high-frequency stimulation. (C, D) Changes in synaptic ultrastructure induced by high-frequency stimulation. (Q A synapse in an uninjected control axon. (D) A synapse within an axon injected with synapsin antibodies. Both axons were from the same spinal cord, which was stimulated at 18 Hz for 6 min immediately prior to hxation. Note the depletion of synaptic vesicles at release sites. Scale bar, 0.2 pm. Modified from Pieribone et al. (1995) Nature d75 493-497, with permission copyright 1995 Macmillan Magazines Ltd.

Results of experiments examining the spatiotemporal dynamics of DNA replication and transcription sites in one-cell embryos were also consistent with the presence of replication-dependent and replication-independent genes (Bouniol-Baly et al., 1997). DNA replication sites were detected by incorporation of digitoxin-modified dUTP (detected with antibodies to digitoxin) and transcription sites were detected by incorporation of BrUTP (detected with anti-BrdU antibodies) following microinjection of both of these nucleotides. While most of the transcription sites did not co-localize with replication sites, there were sites of colocalization. 

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. 

Effect of microinjected antibodies on cell intoxication by Pseudomonas and Shiga toxin. 

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