Recycling embosoms (REs) are transient intermediates of endosomal network, constantly generated from early/sorting endosomes (EEs/SEs). Conventionally, these organelles function in recycling of many growth/nutrient/signalling receptors from SEs to the cell surface and maintain the cellular homeostasis in all cell types. Recent studies have shown that REs slightly diverted their function in specialized cells such as melanocytes for the delivery of melanogenic cargo to a set of lysosome-related organelles (LROs) called melanosomes. However, it is unknown how melanocytes modulate the trafficking routes of REs towards the biogenesis of melanosomes. Any alterations in this process result in occulocutaneous albinism, commonly observed in autosomal recessive disorder, Hermansky-Pudlak Syndrome (HPS). HPS is caused by mutations in nine genes in human and fifteen genes in mouse and the protein products of these genes were grouped in multiple endosomal protein complexes; BLOC (Biogenesis of Lysosome-related Organelles Complex)-1, -2, -3, AP (Adaptor Protein)-3 and HOPS (homotypic fusion and protein sorting). Studies from our laboratory and others have shown that REs deliver the melanin-synthesizing enzymes to melanosome in BLOC-1 and BLOC-2 dependent manner. On the other side, studies in fibroblasts have shown that the adaptor AP-1 and microtubule-dependent motor, KIF13A also regulates the formation of REs. In these studies, it was proposed that AP-1 binds to the cargo tails and interacts with motor KIF13A to generate the RE tubules, where BLOC-1 initiates the biogenesis. Nevertheless, the mechanism behind the biogenesis of REs and how these molecules synergistically control these processes is largely unknown. Additionally, the role of BLOC-2 in REs biogenesis never been implicated. Here we have attempted to study the mechanism of RE biogenesis and their role in pigment granule formation using HeLa and mouse melanocytes as model systems. In general, Rab GTPases (Rabs) regulate the several process of membrane trafficking including cargo sorting, membrane domain organization, tethering and fusion. We hypothesized that the biogenesis of RE is also regulated by one of the endosome localized Rab GTPases. Our RNAi screening against Rabs involved in regulating the RE length/number showed Rab22A as a potential candidate. Thus, we aim to study the role of Rab22A in RE biogenesis and its regulation in melanocyte pigmentation. The current study entitled as “Understanding the role of Rab22A in recycling endosome biogenesis and melanocyte pigmentation” is divided into five chapters. Chapter-I outlines the review of literature on cell biology of intracellular organelles such as endocytic network and melanosomes. Chapter-II details the experimental procedures used in the study. Chapter-III to Chapter-V describes the results and discussion. Chapter-III: Identification of endosomal Rab GTPases required for the dynamics of recycling endosomes Endosomal Rabs are known to regulate various functions such as vesicle biogenesis, transport, tethering and fusion, but their role in generation of tubulo-vesicular carriers of endocytic system, REs is unknown. It has been shown that REs possibly derived from EEs/SEs and characterized by the association/localization of multiple proteins such as transferrin receptor (TfR), SNARE STX13, Rab11 and motor KIF13A. In this study, we have used YFP-KIF13A as a marker to label the REs. YFP-KIF13A in HeLa cells localized to long tubular structures throughout the cell and also to the clusters of peripheral endosomes. To identify the endosomal Rabs that regulate the RE dynamics (both length and number), we have transfected the HeLa cells with shRNA against endosomal Rabs such as Rab4A, Rab5A, Rab5B, Rab5C, Rab7A, Rab9A, Rab11A, Rab14A and Rab22A. Post transfection and shRNA selection, cells were transfected with YFP-KIF13A, analyzed and quantified the RE dynamics using ImageJ. Here, we have measured two parameters for the identification of Rab/s that potentially regulates the REs biogenesis: first, average number of tubules per cell and second, average length of tubules per cell. These studies identified Rab22A as a potential candidate, depletion of this Rab affects both number and average length of KIF13A-positive tubules. As described above, REs deliver several melanocyte specific cargoes to melanosomes in melanocytes. However, the function of Rab22A in controlling these transport steps to melanosome/its biogenesis or pigmentation has not been addressed. Thus, we have studied the mechanism of Rab22A in RE biogenesis and its role in pigmentation in the following sections. Chapter-IV: Characterization of Rab22A function in regulating the recycling endosomes Initially, we tested whether Rab22A localizes to the REs. Our co-expression studies show that Rab22A localizes to KIF13A- or STX13-positive RE compartments in HeLa or melanocytes, respectively. In general, Rab GTPases mediate their function through cycling between GTP (membrane bound) and GDP (cytosol) bound state. These states can be achieved by point mutation of active site residues in the protein. We have generated Rab22A constitutive active mutant (Rab22AQ64L, defective in GTP hydrolysis) and dominant negative mutant (Rab22AS19N, defective in GTP binding) to understand the role of Rab22A in regulating REs. Interestingly, overexpression of Rab22AQ64L mutant in HeLa cells increases the average number of KIF13A-positive REs relative to the wild-type Rab22A (Rab22AWT). As predicted, overexpression of Rab22AS19N mutant reduces the number as well as length of RE tubules relative to the control HeLa cells. Consistent to these studies, Rab22A-knockdown did not affect the endogenous KIF13A protein levels or its recruitment to endosomes, however recycling of TfR (measured through Tf-Alexa 594) was significantly affected in these cells. These studies suggest that Rab22A possibly regulates the formation or function of REs. Likewise, overexpression of Rab22AQ64L and Rab22AS19N mutants in melanocytes resulted in reduction of total melanin content in the cells. To confirm these results, we have performed immunofluorescence microscopy (IFM) analysis, which showed Rab22AQ64L localized to the enlarged vacuolar structures, positive for melanosomal cargo TYRP1 (tyrosinase-related protein 1), whereas Rab22AS19N localized to the cytosol. Further, Rab22A depletion in melanocytes causes the hypopigmentation in the cells concurrently reduces the stability of TYRP1 but not other melanocyte specific proteins, indicating a role for Rab22A in regulating TYRP1 transport to melanosomes. Altogether, our studies suggests that Rab22A regulates the TfR recycling in HeLa cells and TYRP1 transport in melanocytes by controlling the RE dynamics. Chapter-V: Molecular mechanism of recycling endosome biogenesis: a role for Rab22A Rabs perform their function by recruiting specific effector/s to the membrane upon Rab activation. It is unknown, how Rab22A regulates REs through its effectors. We hypothesize that Rab22A may regulate the recruitment and function of BLOC-1 and BLOC-2 complexes during RE formation. To validate these hypothesis, we carried out the knockdown of individual BLOC-1 and -2 subunits (destabilize the entire complex) separately in HeLa and studied the dynamics of RE through YFP-KIF13A expression. As expected, the length and number of KIF13A-postive tubules were significantly reduced in both BLOC-1- and BLOC-2-deficient HeLa cells and was phenocopying the Rab22A knockdown cells. Moreover, subcellular fractionation in HeLa, co-fractionated Rab22A with BLOC-1 (Muted) or BLOC-2 (HPS6) subunits along with KIF13A. Additionally, endogenous subunit levels of BLOC-1 and BLOC-2 were moderately reduced in Rab22A knockdown HeLa cells. Consistent to these results, recycling kinetics of Transferrin (Tf) was altered in Rab22A depleted cells as similar to BLOC-1- or BLOC-2-deficient cells as reported earlier. Likewise, Rab22A knockdown in melanocytes affected STX13-positive tubules and also the stability of endogenous BLOC-1 subunit, Pallidin, suggesting that Rab22A possibly works with BLOC-1 and BLOC-2 independent of cell types. To understand the regulation among these molecules, we overexpressed Rab22A in BLOC-1-deficient cells and analyzed the cells for BLOC-1-deficient rescue phenotypes such as pigmentation and cargo localization. However, Rab22A could not compensate the BLOC-1 function, suggesting that Rab22A possibly functions upstream of BLOC-1. Our subcellular and membrane associated fractionation studies of homogenates depleted with Rab22A, BLOC-1 and BLOC-2 showed that subunit levels of BLOC-1 and BLOC-2 in the membrane pool were significantly reduced upon Rab22A depletion compared to control cells. However, membrane association of Rab22A in BLOC-1 deficient cells was not affected. Further, our biochemical interaction studies showed that Rab22A interacts physically with BLOC-1 and BLOC-2 subunits as well as with KIF13A. Thus, these studies indicate that Rab22A possibly recruits and interacts with BLOC-1 and BLOC-2 for the generation of REs. We have summarized the study by proposing a model wherein Rab22A localizes to the limiting membrane of endosomes that are positive for KIF13A and then recruits and associates with BLOC-1 and BLOC-2 complexes which subsequently pulled by KIF13A for the generation of RE tubules.

The electro–mechanical connection between under bump metallization (UBM) and solder in flip–chip bonding is achieved by the formation of brittle intermetallic compounds (IMCs) during the soldering process. These IMCs continue to grow in the solid–state during storage at room temperature and service at an elevated temperature leading to degradation of the contacts. In this thesis, the diffusion–controlled growth mechanism of the phases and the formation of the Kirkendall voids at the interface of UBM (Cu, Ni, Au, Pd, Pt) and Sn (bulk/electroplated) are studied extensively. Based on the microstructural analysis in SEM and TEM, the presence of bifurcation of the Kirkendall marker plane, a very special phenomenon discovered recently, is found in the Cu–Sn system. The estimated diffusion coefficients at these marker planes indicate one of the reasons for the growth of the Kirkendall voids, which is one of the major reliability concerns in a microelectronic component. Systematic experiments using different purity of Cu are conducted to understand the effect of impurities on the growth of the Kirkendall voids. It is conclusively shown that increase in impurity enhances the growth of voids. The growth rates of the interdiffusion zone are found to be comparable in the Cu–Sn and the Ni–Sn systems. EPMA and TEM analyses indicate the growth of a metastable phase in the Ni–Sn system in the low temperature range. Following, the role of Ni addition in Cu on the growth of IMCs in the Cu–Sn system is studied based on the quantitative diffusion analysis. The analysis of thermodynamic driving forces, microstructure and crystal structure of Cu6Sn5 shed light on the atomic mechanism of diffusion. It does not change the crystal structure of phases; however, the microstructural evolution, the diffusion rates of components and the growth of the Kirkendall voids are strongly influenced in the presence of Ni. Considering microstructure of the product phases in various Cu/Sn and Cu(Ni)/Sn diffusion couples, it has been observed that (i) phases have smaller grains and nucleate repeatedly, when they grow from Cu or Cu(Ni) alloy, and (ii) the same phases have elongated grains, when they grow from another phase. A difference in growth rate of the phases is found in bulk and electroplated diffusion couples in the Au–Sn system. The is explained in AuSn4 based on the estimated tracer diffusion coefficients, homologous temperature of the experiments, grain size distribution and crystal structure of the phase. The growth rates of the phases in the Au–Sn system are compared with the Pd–Sn and the Pt–Sn systems. Similar to the Au–Sn system, the growth rate of the interdiffusion zone is found to be parabolic in the Pd–Sn system; however, it is linear in the Pt–Sn system. Following, the effect of addition of Au, Pd and Pt in Cu is studied on growth rate of the phases. An analysis on the formation of the Kirkendall voids indicates that the addition of Pd or Pt is deleterious to the structure compared to the addition of Au. This study indicates that formation of voids is equally influenced by the presence of inorganic as well as organic impurities.