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Abstract for Live Imaging and Protein Subcellular Localization
Using a Multifunctional Reporter Protein in Mammalian Cell Lines and
Human Neural Progenitors
Soshana Svendsen1, Erin McMillan2, Chad Zimprich1, Elizabeth
Capowski2, Mark McDougall3, Dieter Klaubert3, Clive N. Svendsen2,
Georgyi V. Los1
1Research and Development, Promega Corporation, Madison, WI 53711
2Waisman Center, University of Wisconsin, Madison, WI 53705
3Promega Biosciences, Inc., San Luis Obispo, CA 93401
We demonstrate that the HaloTag® Protein Reporter can be fused
to a truncated human integrin β1 subunit (trβ1IntHT2™) at the amino
terminus for extracellular localization. We used a non-permeable
green fluorophore to distinguish the cell surface-expressed pool for
studying protein internalization and a separate permeable red
fluorophore to distinguish the internal pool for studying protein
trafficking. With both spatial and temporal separation of protein
pools in live cells, we can simultaneously show that the
trβ1IntHT2™ fusion protein is properly trafficked to and from the
plasma membrane. In addition, labeling the trβ1IntHT2™ protein in
live cells followed by fixation and immunocytochemistry for
different subcellular markers shows that the fusion protein
distributes normally with protein trafficking compartments (e.g., ER
and golgi) and protein recycling machinery (e.g., endosomes) and
also shows that the fusion protein retains some expected protein
co-localizations.
We further show that the HaloTag® Protein and fusion proteins
can be expressed in human neural progenitor cells and can be labeled
with different fluorophores. The HaloTag® Protein is well tolerated
and expressed in the expected pattern by human neural progenitor
cells. These results indicate that the HaloTag® Technology is a
useful tool for understanding protein trafficking, recycling, and
subcellular distribution in an array of mammalian cells, including
human neural progenitor cells. |
