Authors: Maya Kardouh, Julia Aguade Gorgorio, Iman Fares, and Hanna Mikkola.
The finding that MYCT1 is downregulated in hematopoietic stem cells (HSCs) and required for their engraftment in immunodeficient mice, as determined by the Mikkola lab, raises a crucial question about how MYCT1 contributes to the “stemness” of HSCs. Our data so far suggest a predominant localization of MYCT1 in the membrane fraction of hematopoietic cells, and our immunofluorescence results suggested the possibility of MYCT1 localizing in vesicles. We also found that the extracellular domain of MYCT1 is required for its subcellular localization. Thus, we asked if MYCT1 localizes in specific type of vesicles that can point to its function, how the deletion of the extracellular domain affects the potential localization of MYCT1 in trafficking vesicles, and what cellular programs MYCT1 may be involved in in HSCs.
A previous publication showed that MYCT1 is localized in cytoplasmic membrane-associated vesicles in Hela cells, where it colocalizes with Clathrin H and Syntaxin 6 (t-SNARE) coated vesicles (Wu et al., 2016). Thus, we will assess MYCT1 colocalization with Syntaxin 6, and other vesicular trafficking candidates such as, EEA1, Rab 5A, APPL1, which are associated with endosomes, Clathrin Heavy Chain (coating protein), Caveolin-1 (membrane protein associated with endocytosis), and GOPC (associated with Golgi). It has been shown that Clathrin mediated endocytosis can regulate signaling programs essential for HSCs (Rai et al., 2014). For example, clathrin assembly lymphoid myeloid leukemia protein (CALM) was shown to have a function in KIT signaling in mouse HSCs (Rai et al., 2014). KIT is a type of receptor tyrosine kinase which is internalized to the early endosome upon ligand binding (Rai et al., 2014). Additionally, while in the early endosome, KIT was shown to activate downstream molecules in mouse HSCs (Rai et al., 2014). Given these findings, if we see colocalization between MYCT1 and any of our selected proteins, we can hypothesize its mechanistic role in hematopoietic cells according to the function of the proteins it colocalizes with. We will also consider how the colocalization of MYCT1 with its extracellular domain deleted compares to the colocalization of the full-length MYCT1 and the selected proteins.
In another recent study, G-protein–coupled receptor–associated sorting proteins (GPRASP1) and GPRASP2, were found to be negative regulators of HSC transplantation (Morales-Hernández et al, 2020). Knockdown of GPRASP1 and GPRASP2 increased the surface levels CXCR4, a master regulator of HSCs transplantation, and prevented its degradation by GPRASP1 and GPRASP2 (Morales-Hernández et al, 2020). Even though there is no evidence that MYCT1 is a negative regulator of HSCs since it is required for HSC expansion and engraftment, it can be operating in a similar fashion and controlling other positive regulators of HSC expansion and engraftment. After performing the trafficking protein co-localization experiment, we will gain insight to possible pathways for MYCT1. Although little is known about MYCT1 function, we are getting closer to unravelling its role in distinct cellular pathways that may prove to be necessary for human HSC expansion and transplantability.
References:
Morales-Hernández, Antonio et al. “GPRASP proteins are critical negative regulators of
hematopoietic stem cell transplantation.” Blood vol. 135,14 (2020): 1111-1123. doi:10.1182/blood.2019003435
Rai, Shinya et al. “Clathrin assembly protein CALM plays a critical role in KIT signaling by regulating its cellular transport from early to late endosomes in hematopoietic cells.”
PloS one vol. 9,10 e109441. 3 Oct. 2014, doi:10.1371/journal.pone.0109441 Wu, Shuai et al. “Transmembrane domain is crucial to the subcellular localization and function of Myc target 1.” Journal of cellular and molecular medicine vol. 20,3 (2016): 471-81 doi:10.1111/jcmm.12747
Picture: https://fineartamerica.com/featured/bone-marrow-transplant-steve-gschmeissner.html? product=canvas-print
A previous publication showed that MYCT1 is localized in cytoplasmic membrane-associated vesicles in Hela cells, where it colocalizes with Clathrin H and Syntaxin 6 (t-SNARE) coated vesicles (Wu et al., 2016). Thus, we will assess MYCT1 colocalization with Syntaxin 6, and other vesicular trafficking candidates such as, EEA1, Rab 5A, APPL1, which are associated with endosomes, Clathrin Heavy Chain (coating protein), Caveolin-1 (membrane protein associated with endocytosis), and GOPC (associated with Golgi). It has been shown that Clathrin mediated endocytosis can regulate signaling programs essential for HSCs (Rai et al., 2014). For example, clathrin assembly lymphoid myeloid leukemia protein (CALM) was shown to have a function in KIT signaling in mouse HSCs (Rai et al., 2014). KIT is a type of receptor tyrosine kinase which is internalized to the early endosome upon ligand binding (Rai et al., 2014). Additionally, while in the early endosome, KIT was shown to activate downstream molecules in mouse HSCs (Rai et al., 2014). Given these findings, if we see colocalization between MYCT1 and any of our selected proteins, we can hypothesize its mechanistic role in hematopoietic cells according to the function of the proteins it colocalizes with. We will also consider how the colocalization of MYCT1 with its extracellular domain deleted compares to the colocalization of the full-length MYCT1 and the selected proteins.
In another recent study, G-protein–coupled receptor–associated sorting proteins (GPRASP1) and GPRASP2, were found to be negative regulators of HSC transplantation (Morales-Hernández et al, 2020). Knockdown of GPRASP1 and GPRASP2 increased the surface levels CXCR4, a master regulator of HSCs transplantation, and prevented its degradation by GPRASP1 and GPRASP2 (Morales-Hernández et al, 2020). Even though there is no evidence that MYCT1 is a negative regulator of HSCs since it is required for HSC expansion and engraftment, it can be operating in a similar fashion and controlling other positive regulators of HSC expansion and engraftment. After performing the trafficking protein co-localization experiment, we will gain insight to possible pathways for MYCT1. Although little is known about MYCT1 function, we are getting closer to unravelling its role in distinct cellular pathways that may prove to be necessary for human HSC expansion and transplantability.
References:
Morales-Hernández, Antonio et al. “GPRASP proteins are critical negative regulators of
hematopoietic stem cell transplantation.” Blood vol. 135,14 (2020): 1111-1123. doi:10.1182/blood.2019003435
Rai, Shinya et al. “Clathrin assembly protein CALM plays a critical role in KIT signaling by regulating its cellular transport from early to late endosomes in hematopoietic cells.”
PloS one vol. 9,10 e109441. 3 Oct. 2014, doi:10.1371/journal.pone.0109441 Wu, Shuai et al. “Transmembrane domain is crucial to the subcellular localization and function of Myc target 1.” Journal of cellular and molecular medicine vol. 20,3 (2016): 471-81 doi:10.1111/jcmm.12747
Picture: https://fineartamerica.com/featured/bone-marrow-transplant-steve-gschmeissner.html? product=canvas-print
Proudly powered by Weebly