AUTHOR=Leek Ashley N. , Quinn Josiah A. , Krapf Diego , Tamkun Michael M.
TITLE=GLT-1a glutamate transporter nanocluster localization is associated with astrocytic actin and neuronal Kv2 clusters at sites of neuron-astrocyte contact
JOURNAL=Frontiers in Cell and Developmental Biology
VOLUME=12
YEAR=2024
URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2024.1334861
DOI=10.3389/fcell.2024.1334861
ISSN=2296-634X
ABSTRACT=Introduction: Astrocytic GLT-1 glutamate transporters ensure the fidelity of glutamic neurotransmission by spatially and temporally limiting glutamate signals. The ability to limit neuronal hyperactivity relies on the localization and diffusion of GLT-1 on the astrocytic surface, however, little is known about the underlying mechanisms. We show that two isoforms of GLT-1, GLT-1a and GLT-1b, form nanoclusters on the surface of transfected astrocytes and HEK-293 cells.
Methods: We used both fixed and live cell super-resolution imaging of fluorescent protein and epitope tagged proteins in co-cultures of rat astrocytes and neurons. Immunofluorescence techniques were also used. GLT1 diffusion was assessed via single particle tracking and fluorescence recovery after photobleach (FRAP).
Results: We found GLT-1a, but not GLT-1b, nanoclusters concentrated adjacent to actin filaments which was maintained after addition of glutamate. GLT-1a nanocluster concentration near actin filaments was prevented by expression of a cytosolic GLT-1a C-terminus, suggesting the C-terminus is involved in the localization adjacent to cortical actin. Using super-resolution imaging, we show that astrocytic GLT-1a and actin co-localize in net-like structures around neuronal Kv2.1 clusters at points of neuron/astrocyte contact.
Conclusion: Overall, these data describe a novel relationship between GLT-1a and cortical actin filaments, which localizes GLT-1a near neuronal structures responsive to ischemic insult.