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Published on: May 12, 2018
Imaging the dynamics of intracellular protein translocation by photoconversion of phamret-cybr/ROM
1Australian Centre for Microscopy and Microanalysis (ACMM), Australian Microscopy and Microanalysis Research Facility (AMMRF), University of Sydney, Australia.
Abstract:
Cybr/Reduced On-random Motile (ROM) is a scaffold protein, containing a postsynaptic density protein-95/discs-large/ZO-1 (PDZ) domain, a LEU region and a PDZ domain binding region at the C-terminus. In the immune system, Cybr/ROM was found to localize in vesicles and at the plasma membrane, through interactions with cytohesin-1. In this investigation, we reported Cybr/ROM as occurring in vesicles, the cytoplasm and at membrane ruffles of H1299 lung cancer cells. Its localization at the ruffles was dependent on intact actin structures as indicated by latrunculin A treatment, which abrogated ruffle formation and staining of Cybr/ROM at the cells' periphery. Transfection of truncation mutants consisting of either the PDZ or LEU domain showed that the LEU domain of ROM was localized to membrane ruffles, vesicles and the cytoplasm, whereas, the PDZ domain localized to the membrane ruffles and cytoplasm only. There was therefore, domain/molecular segregation of Cybr/ROM in different cellular compartments. Cybr/ROM was subcloned into a plasmid carrying the photoactivation-mediated resonance energy transfer (Phamret) protein. The photoconversion experiments demonstrated the diffusion of ROM from the cytoplasm to the membrane ruffling sites and conversely from membrane ruffles to the cytoplasm. Large variances in the transport velocity of Cybr/ROM in the cytoplasm suggested that its movements were facilitated by other mechanisms in addition to diffusion.
Insights
Reduced On-random Motile (ROM) protein localizes to cellular compartments in lung cancer cells. Its movement between cytoplasm and membrane ruffles is actin-dependent and involves diffusion, suggesting additional transport mechanisms.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Cybr/Reduced On-random Motile (ROM) is a scaffold protein with a PDZ domain.
- ROM interacts with cytohesin-1 and localizes to vesicles and plasma membrane in immune cells.
Purpose of the Study:
- To investigate the cellular localization and dynamics of Cybr/ROM in H1299 lung cancer cells.
- To determine the role of specific ROM domains in its subcellular localization.
- To explore the movement mechanisms of ROM within lung cancer cells.
Main Methods:
- Cell culture of H1299 lung cancer cells.
- Treatment with latrunculin A to disrupt actin structures.
- Transfection with ROM domain truncation mutants.
- Subcellular localization studies using microscopy.
- Photoactivation-mediated resonance energy transfer (Phamret) for photoconversion experiments.
Main Results:
- Cybr/ROM localizes to vesicles, cytoplasm, and membrane ruffles in H1299 cells.
- Actin integrity is crucial for ROM localization at membrane ruffles.
- The LEU domain mediates localization to ruffles, vesicles, and cytoplasm.
- The PDZ domain localizes to ruffles and cytoplasm.
- ROM exhibits bidirectional diffusion between cytoplasm and membrane ruffles, with variable transport velocities.
Conclusions:
- Cybr/ROM displays domain-specific compartmentalization within lung cancer cells.
- Actin dynamics regulate ROM's peripheral localization.
- ROM transport involves diffusion and potentially other facilitated mechanisms.
