Reagents

Reagents for studying SNAREs and their function

We have generated a large panel of antibodies and expression constructs for studying the localisation and trafficking of post-Golgi SNAREs. In addition, we have also validated a large number of commercial antibodies over the years. We have generally found that SNARE antibodies from Synaptic Systems and CST are on the whole very good. We also have an increasing number of cell lines which are either deficient or mutant for various SNAREs (VAMPs 3, 4, 8 and SNAP29) which we are happy to share.

SNARE antibodies

HA-tagged gamma-retroviral expression constructs

It is not straightforward to study the trafficking and function of SNAREs as their overexpression leads to drastic changes in intraceullar trafficking. To overcome some of these issues we have used MMLV based expressions systems to generate stable pools of cells. We have generally found that tagged SNAREs are expressed at similar levels to the endogenous SNAREs. As SNARES do not have luminal domains we have generated a panel of constructs where we have introduced a tag at their C-terminus. This tag allows the trafficking and cell surface levels of the proteins to be determined using antibody feeding experiments. We used this method to show that PI-CALM plays an important role in the endocytocis of VAMPs 2, 3 and 8. At present it is still unclear whether these C-terminally tagged SNAREs are fully functional. However, they are able to recapitulate many aspects of the SNAREs trafficking. We have also tried introducing a tag at the C-terminus of Q-SNAREs. However, for the ones we tested do not traffic correctly. Thus, we have tagged the Q-SNAREs at their N-terminus.

Tools for studying constitutive secretion

We have generated a range of different cell lines which express our pharmacologically regulated secretory reporter. These cells are useful for functional genomic experiments and chemical inhibition studies. We have found that the system work best when stable, clonal cell lines are generated. In mammalian cells the system can be used to follow secretion by microscopy and flow cytometry. In drosophila cells you are also able to follow the processing of the cargo biochemically. We are happy to share the cell lines and plasmids for making these cells. We have also recently published a methods chapter which provides a detailed overview of how to make these cells. We have also identified a number of siRNA which are potent inhibitors of constitutive secretion so can be used as positive controls in secretion experiments.

Time course of secretion

The indicated cells lines were induced to secrete the reporter molecule and their fluorescence monitored over time. Approximately 80% of the secretory reporter is secreted in 80 minutes.

Reagents for studying the AP-1 and AP-3 adaptor complexes

Over the years we have generated quite a large number of reagents for studying the function and localisation of adaptor complexes. We are happy to provide these reagents on request.

Mouse monoclonal antibodies against the delta subunit of the AP-3 complex

When I was a postdoc in Richard Scheller's group I generated a panel of mouse monoclonal antibodies to the hinge region of delta subunit of AP-3 complex (608-800aa - validated on fibroblasts derived from mocha mice). SA4 works well for fluorescence microscopy, immunoprecipitations and immuno-electron microscopy. We have good stock of SA4 and KF4 which we are happy to share. You can also get the cells and antibodies from the DSHB. I mapped the epitope which the SA4 antibody binds and the peptide (680-710aa of human δ-adaptin) can be used to isolate native AP-3 complexes. Using this approach in collaboration with Victor Faundez we isolated Phosphatidylinositol 4-Kinase Type II α as novel interaction partner of the AP-3 complex.

AP-3 deficient fibrobalsts and gamma-retroviral based rescue plasmids

As a PhD student in Scottie's lab I generated fibrobalsts from the mocha mouse which is deficient for the delta subunit of the AP-3 complex. These fibroblasts can be very easily reconstituted so are a useful tools for studying trafficking defects cause by the loss of AP-3. The cells do not transfect very efficiently but transduce well using MMLV based systems. The cells are available from the Eurpoean Cell Culture Collection or ATCC. We used these system extensively to study the role of the AP-3 complex on trafficking VAMP7 in collaboration with David Owen and Paul Luzio.

AP-1 complexes can be affinity isolated using mab100.3

While I was working out how to affinity isolate AP-3 complexes I also did something similar for AP-1. Mab100.3 is a mouse monoclonal antibody that does not recognise gamma adaptin from rodents. Thus, it was relatively easy to identify which residues the antibody recognises (661-674aa of human gamma adaptin). Using this approach we identified p200 as a novel interactor for the AP-1 complex. However, as Scottie had got there first so we never pursed this interaction and did not publish the method.

Page updated

Report abuse