Santiago M. Di Pietro

Platelet α-granules are lysosome-related organelles produced in megakaryocytes, the platelet precursor cells. The biogenesis of α-granules is incompletely understood but depends on common endosomal pathways. Here, we discovered GRIPAP1, a partially characterized ubiquitous protein, as a new component of the α-granule biogenesis machinery. GRIPAP1-deficient megakaryocytes showed a significant decrease of α-granule numbers and overall cargo levels. In WT megakaryocytes, fibrinogen taken up by endocytosis and newly synthesized PF4 trafficked through GRIPAP1-labeled compartments en route to α-granules. GRIPAP1 localized to endosome subdomains decorated by Rab4a and Stx12, known players in α-granule biogenesis. GRIPAP1 bound GTP-loaded Rab4a, a key interaction for GRIPAP1 recruitment to membranes. Biochemically, GRIPAP1 behaved as an elongated homodimer akin to membrane tethering factors. Consistently, artificial mislocalization of GRIPAP1 to the mitochondria was sufficient to recruit Rab4a compartments containing internalized transferrin and newly synthesized PF4 to mitochondria. Together, the data advance understanding of endosomal transport, the biogenesis of α-granules, and likely other endo-lysosomal organelles.

The endosomal Commander assembly associates with the sorting nexin-17 (SNX17) cargo adaptor to regulate cell surface recycling of internalised integral proteins including integrins and lipoprotein receptors. Here, we identify leucine rich melanocyte differentiation associated (LRMDA) as a Commander binding protein. We reveal that LRMDA and SNX17 share a common mechanism of Commander association, and that LRMDA simultaneously associates with Commander and active RAB32, establishing distinct RAB32-LRMDA-Commander and SNX17-Commander assemblies. Functional analysis in melanocytes reveals distinct roles for RAB32-LRMDA-Commander and SNX17-Commander in melanosome biogenesis. We reveal how LRMDA mutations, causative for oculocutaneous albinism type 7, a hypopigmentation disorder accompanied by poor visual acuity, uncouple RAB32 and Commander binding thereby establishing the mechanistic basis of this disease. Our discovery of this alternative Commander assembly highlights the plasticity of Commander function in human pigmentation and extends the Commander function beyond the SNX17-mediated regulation of cell surface proteome.

Platelets are anucleate cellular fragments derived from megakaryocytes (MKs) and α-granules constitute their most numerous membrane-bound compartments. These granules play a role in platelet aggregation to form a hemostatic plug but also contain numerous cargo proteins with key functions in angiogenesis, inflammation, wound healing and cancer. Human genetic disorders that cause deficiencies in the biogenesis of platelet α-granules manifest with prolonged bleeding. The initial studies on platelets and MKs from these patients provided a first glimpse into the biosynthesis of α-granules as a membrane trafficking problem. Significant progress in the field has been made in recent years in part due to the creation of iPSC-derived megakaryocytic cells capable of releasing functional platelets, thus overcoming the limitations of working with primary MKs. The emerging model indicates that sorting and recycling endosomes are key intermediate stations traversed by α-granule cargo on their way to the α-granule. Here we describe the different trafficking pathways used by α-granule proteins and elaborate on their commonalities. Similar to other lysosome-related organelles, most of the proteins involved in the biogenesis of α-granules are ubiquitously expressed and we discuss NBEAL2 as a factor highly expressed in MKs that likely diverts this machinery to make α-granules. Importantly, understanding the trafficking pathways involved in the making of the α-granule has an impact not only on platelet biology but may also illuminate the broader lysosome-related organelle field.

Clathrin-mediated endocytosis depends on polymerization of a branched actin network to provide force for membrane invagination. A key regulator in branched actin network formation is actin capping protein (CP), which binds to the barbed end of actin filaments to prevent the addition or loss of actin subunits. CP was thought to stochastically bind actin filaments, but recent evidence shows CP is regulated by a group of proteins containing CP-interacting (CPI) motifs. Importantly, how CPI motif proteins function together to regulate CP is poorly understood. Here, we show Aim21 and Bsp1 work synergistically to recruit CP to the endocytic actin network in budding yeast through their CPI motifs, which also allosterically modulate capping strength. In contrast, twinfilin works downstream of CP recruitment, regulating the turnover of CP through its CPI motif and a non-allosteric mechanism. Collectively, our findings reveal how three CPI motif proteins work together to regulate CP in a stepwise fashion during endocytosis.

Point mutations in LRRK2 cause Parkinson’s Disease and augment LRRK2’s kinase activity. However, cellular pathways that enhance LRRK2 kinase function have not been identified. While overexpressed Rab29 draws LRRK2 to Golgi membranes to increase LRRK2 kinase activity, there is little evidence that endogenous Rab29 performs this function under physiological conditions. Here we identify Rab38 as a novel physiological regulator of LRRK2. In mouse melanocytes, which express high levels of Rab38, Rab32, and Rab29, knockdown of Rab38 but not Rab32 or Rab29 decreases phosphorylation of multiple LRRK2 substrates, including Rab10 and Rab12, by both exogenous and endogenous LRRK2. In B16-F10 mouse melanoma cells, Rab38 drives LRRK2 membrane association, and overexpressed kinase-active but not kinase-inactive LRRK2 shows striking pericentriolar recruitment, which is dependent on the presence of endogenous Rab38 but not Rab32 or Rab29. Deletion or mutation of LRRK2 at the Rab38 binding site in the N-terminal armadillo domain decreases LRRK2 membrane association, pericentriolar recruitment, and ability to phosphorylate Rab10. Consistently, overexpression of LRRK2 _350-550 , a fragment that encompasses the Rab38 binding site, blocks endogenous LRRK2’s phosphorylation of Thr73-Rab10. Finally, disruption of BLOC-3, the guanine nucleotide exchange factor for Rab38 and 32, inhibits Rab38’s regulation of LRRK2. In sum, our data identify Rab38 as a physiologic regulator of LRRK2 function and lend support to a model in which LRRK2 plays a central role in Rab GTPase coordination of vesicular trafficking.

Platelet α-granules regulate hemostasis and myriad other physiological processes, but their biogenesis is unclear. Mutations in only 3 proteins are known to cause α-granule defects and bleeding disorders in humans. Two such proteins, VPS16B and VPS33B, form a complex mediating transport of newly synthesized α-granule proteins through megakaryocyte (MK) endosomal compartments. It is unclear how the VPS16B/VPS33B complex accomplishes this function. Here we report VPS16B/VPS33B associates physically with Syntaxin 12 (Stx12), a SNARE protein that mediates vesicle fusion at endosomes. Importantly, Stx12-deficient MKs display reduced α-granule numbers and overall levels of α-granule proteins, thus revealing Stx12 as a new component of the α-granule biogenesis machinery. VPS16B/VPS33B also binds CCDC22, a component of the CCC complex working at endosome exit sites. CCDC22 competes with Stx12 for binding to VPS16B/VPS33B, suggesting a possible hand-off mechanism. Moreover, the major CCC form expressed in MKs contains COMMD3, one of 10 COMMD proteins. Deficiency of COMMD3/CCDC22 causes reduced α-granule numbers and overall levels of α-granule proteins, establishing the COMMD3/CCC complex as a new factor in α-granule biogenesis. Furthermore, P-selectin traffics through the cell surface in a COMMD3-dependent manner and depletion of COMMD3 results in lysosomal degradation of P-selectin and PF4. Stx12 and COMMD3/CCC deficiency cause less severe phenotypes than VPS16B/VPS33B deficiency, suggesting Stx12 and COMMD3/CCC assist but are less important than VPS16B/VPS33B in α-granule biogenesis. Mechanistically, our results suggest VPS16B/VPS33B coordinates the endosomal entry and exit of α-granule proteins by linking the fusogenic machinery with a ubiquitous endosomal retrieval complex that is repurposed in MKs to make α-granules.

Clathrin- and actin-mediated endocytosis is a fundamental process in eukaryotic cells. Previously, we discovered Tda2 as a new yeast dynein light chain that works with Aim21 to regulate actin assembly during endocytosis. Here, we show Tda2 functions as a dimerization engine bringing two Aim21 molecules together using a novel binding surface different than the canonical dynein light chain ligand binding groove. Point mutations on either protein that diminish the Tda2-Aim21 interaction in vitro cause the same in vivo phenotype as TDA2 deletion showing reduced actin capping protein recruitment and increased filamentous actin at endocytic sites. Remarkably, chemically induced dimerization of Aim21 rescues the endocytic phenotype of TDA2 deletion. We also uncovered a capping protein interacting motif in Aim21, expanding its function to a fundamental cellular pathway and showing such motif exists outside mammalian cells. Furthermore, specific disruption of this motif causes the same deficit of actin capping protein recruitment and increased filamentous actin at endocytic sites as AIM21 deletion. Thus, the data indicates the Tda2-Aim21 complex functions in actin assembly primarily through capping protein regulation. Collectively, our results provide a mechanistic view of the Tda2-Aim21 complex and its function in actin network regulation at endocytic sites.

Endocytosis of plasma membrane proteins is mediated by their interaction with adaptor proteins. Conversely, emerging evidence suggests adaptor protein recruitment to the plasma membrane may depend on binding to endocytic cargo. To test this idea we analyzed the yeast adaptor protein Sla1, which binds membrane proteins harboring the endocytic signal NPFxD via the Sla1 SHD1 domain. Consistently, SHD1 point mutations that disrupt NPFxD binding caused a proportional reduction of Sla1-GFP recruitment to endocytic sites. Furthermore, simultaneous SHD1 point mutation and deletion of the SR region linking Sla1 to coat proteins Pan1 and End3 resulted in total loss of Sla1-GFP recruitment to the plasma membrane. The data suggests multiple interactions are needed for recruitment to the membrane. Interestingly, a Sla1 fragment containing just the third SH3 domain – which binds ubiquitin – and SHD1, displayed broad surface localization suggesting plasma membrane recruitment is mediated by interaction with both NPFxD-containing and ubiquitinated plasma membrane proteins. Our results also imply that a Sla1 NPF motif adjacent to the SR region may regulate the Sla1-cargo interaction, mechanistically linking Sla1 cargo binding to endocytic site recruitment.