molecular characterization of the caenorhabditis elegans alp/enigma gene alp-1 caroline r. mckeown,...

Molecular characterization of the Caenorhabditis elegans

ALP/Enigma gene alp-1

Caroline R. McKeown, Hsiao-Fen Han, and Mary C. Beckerle

Developmental Dynamics Vol 235, Issue 2

Muscle Attachment Sites

• Found in dense bodies• Crosslinks actin filaments• Defective -actinin results in

cardiomyopathy (not good)

-Actinin

• ALP (-actinin associated LIM protein)• Family includes at least 7 proteins

• ALP (which has subfamilies including ALP, RIL, CLP36/Elfin, and Mystique)

• Enigma

• Cypher

• All bind either -actinin or -tropomyosin and localize to Z-lines

Mammalian ALP

• LIM (LIN-11, Isl-1, MEC-3) • PDZ (PSD95, Discs-large, ZO-1)

• both are known protein binding interfaces

ALP family domains

C. elegans ALP is eat-1

• First uncovered in a screen by Leon Avery for mutants with pharyngeal pumping defects

• somewhat Unc, also has irregular pharyngeal pumping

• SAGE data shows raised expression levels in larval stages

• similar expression pattern for -actinin

C. elegans ALP is eat-1

C. elegans ALP is eat-1

• Made GFP translational fusions for alp-1

ALP-1 Localization

• All isoforms localize to dense bodies

• ALP-1BCD also found at dense plaques• sites of muscle-muscle adhesion at the muscle termini (vinculin co-localization)

ALP-1 Localization

• Sufficient for localization to dense bodies• not sufficient to localize to dense plaques

• Made PDZ domain containing (and myc-tagged) construct under influence of heat shock promoter

PDZ Domain

• Not essential for viability• PDZ domain localizes protein to sites of

-actinin localization• LIM proteins enable additional protein-

protein interactions• many interactions in Y2H interactome project

• More study needed for specific role

Conclusions

Layton getting CRAZY!!!