Date of Graduation


Document Type

Thesis (MS)

Program Affiliation

Biomedical Sciences

Degree Name

Masters of Science (MS)

Advisor/Committee Chair

Michael X. Zhu, Ph.D

Committee Member

Guangwei Du, Ph.D

Committee Member

Dongfang Liu, Ph.D

Committee Member

Kartik Venkatachalam, Ph.D

Committee Member

Neal Waxham


Both TRPML1 and TRPML3 are members of the mucolipin subfamily of Transient Receptor Potential (TRP) channels. They have been implicated in endolysosomal functions such as divalent cation release, luminal pH regulation, autophagy and vesicle trafficking. Interestingly, whereas TRPML1 is almost extensively localized in lysosomes, TRPML3 is typically found in both lysosomes and the plasma membrane (PM). It has been shown that TRPML3 localization depends on TRPML1, but the mechanism remains unknown. Recently, a number of TRPML1 mutants have been reported to be either retained in the endoplasmic reticulum (ER) or preferentially targeted to the PM. Coexpressing these mutants with either wild type TRPML3 or a non-functional ER-retained mutant, TRPML3-KK, I examined how differentially targeted TRPML1 proteins affect TRPML3 localization. I show that while the wild type TRPML1 decreased the PM targeting of TRPML3, the PM-targeted TRPML1 mutants did not enhance PM localization of TRPML3. Interestingly, not only the wild type TRPML1 brought TRPML3-KK to lysosomes, but also TRPML3 brought the ER-retained TRPML1-KK mutant to these acidic organelles. Moreover, coexpression of TRPML-KK not only reduced TRPML3-mediated Ca2+ response to a TRPML agonist, ML-SA1, but also nearly abolished PM localization of TRPML3. Activation of heteromultimeric TRPML1/3 channels by ML-SA1 significantly increased the PM-localized TRPML3, while the TRPML channel inhibitor, ML-SI1, did not decrease the PM-localized TRPML3. Consistently, co-expressing a non-functional lysosome-localized TRPML1 mutant, TRPML1-F465L with TRPML3 decreased the PM-localized TRPML3 as efficiently as wildtype TRPML1. I conclude that while the lysosome targeting sequence(s) is important for sorting TRPML channels out of ER-Golgi network, the ion-conducting function is important for TRPML channels to be trafficked to plasma membrane, but not for their endocytosis.

Genetically encoded fluorescence probes have gained popularity nowadays because of the ease to target them to specific subcellular compartments. GCaMPs are widely used genetically encoded calcium indicators, which exhibit dramatic increases in fluorescence upon binding to Ca2+. In order to assess Ca2+ signals generated from opening of TRPML1 channel, as well as that from lysosomes, I have engineered two constructs: with GCaMP5G fused to either the C-terminus of TRPML1 or that of lysosomal-associated membrane protein 1 (LAMP1). To my surprise, however, both engineered probes were able to sense Ca2+ released from ER and Ca2+ entry through PM. They did not show specific utility in detecting lysosomal Ca2+ release. Trafficking of LAMP1 and TRPML1 were apparently disrupted with the conjugation of GCaMP5G. Specifically, LAMP1-GCaMP5G and TRPML1-GCaMP5G showed prominent PM localization, unlike the GFP-tagged LAMP1 and TRPML1, which preferentially target to the lysosomes. In addition, TRPML1-GCaMP5G also appeared in enlarged vesicles with bright green fluorescence. These results suggest that cautions should be taken when using engineered GCaMPs to study subcellular localized Ca2+ signals using tagged proteins.


protein trafficking, lysosomal calcium release, TRP channel, lysosome exocytosis, genetic calcium indicator