1.4.1 A IMS OF MY S TUDY
In the past, proteins and mechanisms involved in IHC exocytosis have extensively been examined.
However, knowledge about SV recycling in these highly adapted sensory cells remained sparse. Few studies have shown that endocytic mechanisms in IHCs are activity-dependent and contain clathrin-dependent as well as clathrin-inclathrin-dependent pathways (Jung et al., 2015b; Kamin et al., 2014; Moser and Beutner, 2000; Neef et al., 2014; Revelo et al., 2014). Yet, molecular key players in IHC endocytosis are largely unknown. Neither do we currently understand, how exo- and endocytosis are balanced and coupled in IHCs, although deficits in SV replenishment were shown to go along with severe hearing impairments ( et al., 2010; Strenzke et al., 2016). Studies in CNS synapses have not only focused on the characterization of endocytic proteins mediating different steps and mechanisms of SV recycling; they also demonstrated that proteins originally identified as endocytic factors are involved in release site clearance, protein sorting and coupling of different steps within the SV cycle (for review see Maritzen and Haucke, 2018). Endophilin-A has originally been described as hub orchestrating different steps of CME with a rate-limiting function in the uncoating of CCVs (Milosevic et al., 2011). Recently, endophilin-A was additionally shown to act in clathrin-independent endocytosis via regulating the fission of endocytic vesicles during UFE (Watanabe et al., 2018). The adaptor protein AP180 is not only required for the recruitment of clathrin, but has also a function in release site clearance via sorting of VAMP2 (Koo et al., 2015).
In the here presented two studies, I combined a set of advanced imaging techniques to characterize morphological changes in mouse mutants missing different endophilin-A genes or AP180. This way, I coul examine the distinct roles of these two proteins/ protein families in IHCs with a focus on their functions in SV recycling. Combining the two studies, I could compare different endocytic steps being dependent on either of the two proteins. This way, I gained deeper insights into the relevance of the different endocytic mechanisms for IHC synaptic transmission.
1.4.2 E XPERIMENTAL A PPROACH
In the first study presented, I examined a set of mutants lacking different endophilin genes. In detail, I compared Wt mice with mutants missing endophilin-A1 and -A2 (1/2-DKOs) as well as their littermates missing only endophilin-A1 (1-SKOs), all with the same mixed genetic background
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(approx. 80% Bl6/J and 20% S-129). Moreover, I analyzed endophilin A1 and A3 mutants that were heterozygous or wildtype for endophilin A2. Since neither microscopical nor physiological analyses revealed any differences in IHCs if endophilin A2 was heterozygously expressed, we pooled those two mutants as one group of 1/3-DKOs. I used all of these mutants to perform electron microscopy experiments. Due to our interest in the general organization of the AZ including endocytic intermediates, and to guarantee for comparability with a previous study on AP-2µ in IHCs (Jung et al., 2015b), I performed conventional EM embeddings including chemical fixation, partially with preceding 15 min high-K+ stimulation. Like in the AP-2µ study, I mainly performed my analyses on images from random ultrathin sections acquired with 12,000 x magnification (for analysis of RA-SV and MP-SV numbers) and with 5,000 x magnification (to analyze the ribbon proximity within 1 µm radius from the ribbon) on a JEOL JEM1011 electron microscope. I additionally performed electron tomography on 1/2-DKOs and 1/3-DKOs in comparison to Wt. Only with the 3D information of the
acquired tomograms I could m
-observed in absence of dynamins (Ferguson et al., 2007; Ferguson et al., 2009), nor malformed SVs (as e.g. detected in mutants harboring a point mutation within the otoferlin gene (Strenzke et al., 2016)) were present in the endophilin mutants. Moreover, I could reliably analyze the diameter of SVs. Nonetheless, even with the increased resolution and 3D information of tomograms, examinations on ELVs, which can expand for more than 250 nm in each direction (thickness of sections for electron tomography used here), are difficult to interpret. I could for example not always clearly state if an ELV is connected to the plasma membrane or not. Neither was it possible to differentiate between newly formed ELVs and bona fide endosomes based on their morphology.
Therefore, I categorized all large membranous organelles without clathrin-coat as ELVs in my quantifications. I combined my comprehensive electron microscopy analyses with confocal microscopy. Since we observed impairments in the sustained phase of exocytosis in 1/3-DKOs, we were curious if these changes are caused by reduced otoferlin levels. To test this, I used well-established immunostaining protocols and analysis routines (Strenzke et al., 2016). Moreover, I analyzed the area of Ca2+-channel clusters beneath the ribbon from 2D STED microscopy images as previously described for IHC ribbon synapses (Jean et al., 2018; Krinner et al., 2017; Neef et al., 2018), since we observed slightly decreased Ca2+-currents in several endophilin mutants. My collaborators performed electrophysiology, biochemistry and systems physiology experiments to generate a wholesome characterization of the IHC phenotype in absence of the different endophilins.
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For my second study, I performed HPF and freeze substitution combined with electron tomography in Wt and AP180-KO mice. It is commonly accepted, that this method allows for a near-to-native state structural preservation of filamentous and membranous structures. Yet, identifying clathrin-coated structures in high-pressure frozen samples is more challenging than in conventionally
embedded tissue. We used HPF since AP180
was also shown to be involved in release site clearance in neurons (Koo et al., 2015) and we were thus particularly interested in the AZ membrane and tethered SVs. The combination of HPF and electron tomography allows for a thorough analysis of tethers connecting SVs. Variations in tether numbers and tether lengths have previously been studied to morphologically characterize potential defects up- or downstream of docking (Chakrabarti et al., 2018; Vogl et al., 2015). Furthermore, electron tomography allowed me to more reliably quantify ELV volumes and numbers of budding CCPs, both indicators for clathrin-dependent SV reformation. I additionally performed immunohistochemical stainings following standard protocols and using well-established IHC markers. Together, my morphological studies gave new insights also into the potential functions of AP180 in IHCs.
C HAPTER O NE
Chapter One |
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Chapter One | General Information
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