Dense core vesicle biogenesis in the Golgi apparatus of mammalian neurons

The general aim of this thesis was to study proteins and processes involved in DCV biogenesis in mammalian neurons. In chapter 2 we studied the role of the SNARE proteins Vti1a/b. Previous research had shown that Vti1a/b are important for DCV biogenesis and retrograde trafficking, which led to the hypothesis that Vti1a/b are required for retrieval of a DCV sorting receptor to the Golgi apparatus. Therefore, deeper insight into the consequences of loss of Vti1a/b could help to identify requirements for DCV biogenesis and protein sorting. We confirmed that the Golgi apparatus and DCV biogenesis were disrupted in absence of Vti1a/b. Specifically, the cis-/medial Golgi and the TGN were differentially affected. In addition, we observed that the ER and LAMP1 positive vesicles were affected by loss of Vti1a/b. Finally, we showed that disturbing membrane homeostasis results in a similar phenotype, but this cannot be rescued by myriocin or overexpression of SMS1/2. Taken together, Vti1a/b are important for distinct aspects of TGN and cis-/medial Golgi organization and for a normal distribution of proteins involved in regulated secretion. In chapter 3 we characterized the localization of proteins in the Golgi apparatus, by systematically comparing localization of 21 endogenous proteins to a TGN marker. For some proteins, their localization was previously studied using overexpression or different cell types, but localization of endogenous proteins was not yet performed in mammalian neurons. Seven proteins completely overlapped with TGN38 and four proteins were upstream of TGN38, localizing to the cis-/medial Golgi. Proteins downstream of TGN38 were located at varying distances from TGN38. These localizations were consistent with previously described protein functions. Taken together, in this chapter we made an overview of the localization of 21 endogenous proteins in the Golgi apparatus of mammalian neurons. In chapter 4 we investigated the canonical TGN marker TGN38. We previously observed that loss of Vti1a/b has differential effects on TGN38 localization depending on the TGN38 antibody used. To address this discrepancy, we systematically evaluated four TGN38 antibodies. We observed that antibodies against TGN38 raised against different epitopes resulted in different immunostaining and western blotting results. After ruling out several cell biological explanations, we concluded that the two antibodies raised against the cytoplasmic domain of TGN38 recognize one or multiple other peptides, in addition to TGN38. Thus, this should be taken into account when using these antibodies. HID1 has been implicated in the biogenesis of DCVs in several cell types. In chapter 5 we studied the role of HID1 in DCV biogenesis in primary neurons. Upon HID1 depletion using dCas9, DCV puncta intensity was increased. In addition, intensity of both overexpressed and endogenous luminal DCV cargo was increased in the Golgi apparatus, while DCV membrane protein Syt1 was not affected by loss of HID1. Taken together, we showed that HID1 is important for DCV biogenesis in primary mouse neurons.