Mechanical manipulation and characterization of biological cells have wide applications in genetics, reproductive biology, and cell mechanics. This research focuses on (1) the development of enabling microrobotic systems and techniques for automated cell microinjection and in situ mechanical characterization; and (2) the demonstration of molecule efficacy testing and cell quality assessment with the new technologies. Targeting high-speed cell injection for molecule screening, a first-of-its-kind automated microrobotic cell injection system is developed for injecting foreign materials (e.g., DNA, morpholinos, and proteins) into zebrafish embryos (~1.2 millimeter) and mouse oocytes/embryos (~100 micrometers), which overcomes the problems inherent in manual operation, such as long learning curves, human fatigue, and large variations in success rates due to poor reproducibility. Novel cell holding devices are developed for immobilizing a large number of embryos into a regular pattern, greatly facilitating sample preparation and increasing the sample preparation speed. Leveraging motion control and computer vision techniques, the microrobotic system is capable of performing robust cell injection at a high speed with high survival, success, and phenotypic rates. The mouse embryo injection system is applied to molecule testing of recombinant mitochondrial proteins. The efficacy of an anti-apoptotic Bcl-xL (Delta_TM) protein is, for the first time, quantitatively evaluated for enhancing the development competence of mouse embryos. For cell quality assessment, this research develops a vision-based technique for real-time cellular force measurement and in situ mechanical characterization of individual cells during microinjection. A microfabricated elastic device and a sub-pixel computer vision tracking algorithm together resolve cellular forces at the nanonewton level. Experimental results on young and old mouse oocytes demonstrate that the in situ obtained force-deformation data can be used for mechanically distinguishing healthy mouse oocytes from those with cellular dysfunctions. This work represents the first study that quantified the mechanical difference between young and old mouse oocytes, promising a practical way for oocyte quality assessment during microinjection.

Mechanical manipulation and characterization of biological cells have wide applications in genetics, reproductive biology, and cell mechanics. This research focuses on (1) the development of enabling microrobotic systems and techniques for automated cell microinjection and in situ mechanical characterization; and (2) the demonstration of molecule efficacy testing and cell quality assessment with the new technologies. Targeting high-speed cell injection for molecule screening, a first-of-its-kind automated microrobotic cell injection system is developed for injecting foreign materials (e.g., DNA, morpholinos, and proteins) into zebrafish embryos (~1.2 millimeter) and mouse oocytes/embryos (~100 micrometers), which overcomes the problems inherent in manual operation, such as long learning curves, human fatigue, and large variations in success rates due to poor reproducibility. Novel cell holding devices are developed for immobilizing a large number of embryos into a regular pattern, greatly facilitating sample preparation and increasing the sample preparation speed. Leveraging motion control and computer vision techniques, the microrobotic system is capable of performing robust cell injection at a high speed with high survival, success, and phenotypic rates. The mouse embryo injection system is applied to molecule testing of recombinant mitochondrial proteins. The efficacy of an anti-apoptotic Bcl-xL (Delta_TM) protein is, for the first time, quantitatively evaluated for enhancing the development competence of mouse embryos. For cell quality assessment, this research develops a vision-based technique for real-time cellular force measurement and in situ mechanical characterization of individual cells during microinjection. A microfabricated elastic device and a sub-pixel computer vision tracking algorithm together resolve cellular forces at the nanonewton level. Experimental results on young and old mouse oocytes demonstrate that the in situ obtained force-deformation data can be used for mechanically distinguishing healthy mouse oocytes from those with cellular dysfunctions. This work represents the first study that quantified the mechanical difference between young and old mouse oocytes, promising a practical way for oocyte quality assessment during microinjection.

Mitochondria are essential eukaryotic organelles, acting as the sites for numerous crucial metabolic and signalling pathways. The biogenesis of mitochondria requires efficient targeting of several hundreds of proteins from the cytosol, to their varied functional locations within the organelle. The translocation of localized proteins across the inner membrane, and their subsequent folding is achieved by the ATP-dependent function of mitochondrial Hsp70 (mtHsp70). It is a bonafide member of the Hsp70 chaperone family, which are involved in a multitude of functions, together aimed at protein quality control and maintenance of cellular homeostasis. These varied functions of Hsp70 proteins require binding to exposed hydrophobic patches in substrate polypeptides thus preventing non-productive associations. The interaction with substrates occurs through the substrate-binding domain (SBD) and is regulated by the ATPase activity of the nucleotide-binding domain (NBD), through a series of conformational changes. Conversely, substrate binding to the SBD also stimulates ATP hydrolysis, and thereby the core activities of the two domains are regulated by mutual allosteric signalling. This mechanism of bidirectional inter-domain communication is indispensable for Hsp70 function, which is characterized by cycles of substrate binding and release, coupled to cycles of ATP binding and hydrolysis. The process of allosteric regulation in Hsp70 proteins has been comprehensively investigated, especially in the bacterial homolog, DnaK. However, the in vivo functional significance of inter-domain communication in the eukaryotic mtHsp70 system and the mechanism of its regulation remain unexplored. Furthermore, the complex physiological implications of impairment in allosteric communication and their correlation with diverse disease conditions, including Myelodysplastic syndrome (MDS), and Parkinson’s disease (PD), are yet to be elucidated. Based on this brief introduction, the primary research objectives set out in the present thesis were to: 1. uncover the regulation of ligand-modulated allosteric communication between the two domains of mtHsp70; and its in vivo significance in the context of protein import into the organelle. (Chapter 2) 2. understand the role of mtHsp70 in progression of Parkinson’s disease; and to study the modulation of α-synuclein toxicity by the protein quality control function of the mtHsp70 chaperone network. (Chapters 3 and 4) We have employed a battery of genetic and biochemical approaches to investigate the above questions using the Saccharomyces cerevisiae mtHsp70 protein, Ssc1; an essential protein that is involved in a plethora of critical functions in this eukaryotic model system. Objective 1: Structural studies, primarily in bacterial DnaK, have yielded mechanistic insights into its interactions with ligands and cochaperones, as well as conformational transitions in different ligand-bound states. In recent years, the availability of crystal structures of full-length DnaK and detailed information from NMR studies and single-molecule resolution spectroscopic analyses (both DnaK and eukaryotic Hsp70s), have significantly contributed to our understanding of the inter-domain interface, critical residues and contacts, and the energetics of the entire process of ligand-modulated conformational changes. Although eukaryotic mtHsp70s have a high degree of conservation with DnaK, they possess significant differences in their conformational and biochemical properties. They are essential for a vast repertoire of physiological functions, which are distinctly different from their bacterial counterpart. Using a combined in vivo and in vitro approach, we have uncovered specific structural elements within mtHsp70s, which are required for allosteric modulation of the chaperone cycle and maintenance of in vivo functions of the protein. Foremost, we demonstrate that a conserved SBD loop, L4,5 plays a critical role in inter-domain communication, and multiple mutations in this loop result in significant growth and protein translocation defects. The mutants are associated with a specific set of altered biochemical properties, which are indicative of impaired inter-domain communication. Using the loop L4,5 mutant, E467A as a template for genetic screening, we report a series of intragenic suppressor mutations, which are capable of correcting a distinct subset of the altered properties, and thereby leading to restoration of in vivo functions, including growth, preprotein import and mitochondria biogenesis. The suppressors modify the altered conformational landscape associated with E467A, and also provide us with information regarding unique aspects governing the regulation of allosteric communication, especially in physiological contexts. Strikingly, they reveal that restoration of communication in the NBD to SBD direction is sufficient for function, when the protein is primed in a high ATPase activity state. In this unique scenario, the requirement for ATPase stimulation upon substrate binding is rendered unnecessary, thereby making conformational changes in the SBD to NBD direction, dispensable for function. Further, we provide evidence to show that loop L4,5 functions synergistically with the linker region, working in tandem for organization of the inter-domain interface and propagation of communication. Together, our analyses provide the first insights into regulation of allosteric inter-domain communication in vivo and their implications in mitochondrial protein translocation and organelle biogenesis. Objective 2: Point mutations in the loop L4,5 have been associated with Myelodysplastic syndrome. Additionally, a mutation isolated in clinical cases of Parkinson’s disease was found to be impaired in allosteric communication. These observations further highlight the importance of efficient inter-domain communication in mtHsp70 in the complex physiological scenario of eukaryotic cells. Independent clinical screens of PD patients have revealed unique point mutations in the mtHsp70 and a strong association of the gene locus with the disease progression. This is also correlated with decreased mtHsp70 levels in affected neurons and the interactions of this protein with established PD-candidate proteins like α-synuclein and Dj-1. Further, mitochondrial dysfunction is a common phenomenon associated with neurodegenerative disorders. To understand the specific role of mtHsp70 in PD, we have developed a yeast model for studying the disease variants in isolation from other players of the multifactorial disease, and in complete absence of the wild type protein. We generated two analogous PD-mutations in Ssc1, R103W and P486S; which recapitulated the symptoms of mitochondrial dysfunction in affected neurons, including cell death, inner membrane depolarization, increased generation of ROS, and respiratory incompetence. At the molecular level, we observed an increased aggregation propensity of R103W, while P486S exhibited futile enhanced interaction with J-protein cochaperone partners thereby resulting in loss of chaperoning activity and impaired mitochondrial protein quality control. Remarkably, these altered biochemical properties mimicked similar defects in the human mtHsp70 variants, therefore, affirming the involvement of mtHsp70 in PD progression. To further investigate the relevance of impaired mitochondrial protein quality control in PD, we have explored whether mtHsp70 can act as a genetic modifier of α-synuclein toxicity. It is known that α-synuclein can act as an unfolded substrate for the Hsp70 chaperone system and also deposits as intracellular aggregates in PD-affected brains. Intriguingly, it is known to translocate into mitochondria under conditions of neuronal stress in spite of lacking a canonical mitochondrial signal sequence. Utilizing our yeast-PD model, we find that targeting of α-synuclein A30P disease variant into mitochondria leads to a severe mitochondrial dysfunction phenotype in the wild type Ssc1 background, but not the P486S mutant background. This results in multiple cellular manifestations, which are reversed upon overexpression of the Ssc1 chaperone. Significantly, increasing the J-protein cochaperone availability also leads to reversal of the mutant-associated defects. However, the simultaneous overexpression of both together does not additively improve the protective effects; highlighting the importance of the relative availability of chaperone and cochaperone proteins in preventing aggregation. Our analyses further reveal that while both the wild type and P486S Ssc1 proteins are equally capable of delaying aggregation of α-synuclein, only the wild-type chaperone is better able to prevent aggregation in the presence of its J-protein cochaperone, leading to accumulation of soluble oligomeric species. These observations raised the intriguing possibility, that the reduced chaperoning ability of the proline to serine PD-mutant is, in fact, a compensatory adaptation, favoring the aggregation of α-synuclein over its more toxic soluble oligomeric form. We verify this hypothesis with the aggregation kinetics of A30P α-synuclein, whose intrinsically lower aggregation tendency results in a pronounced delay in aggregation with the wild-type chaperone, thereby strongly favoring the toxic oligomeric species and correlating with the observed lethality in yeast cells. In conclusion, our study provides a model of α-synuclein aggregation-related toxicity and its modulation by the extent of protein quality control within the mitochondrial matrix, through the action of the mtHsp70 chaperone network.

Maintenance of genomic integrity and stability is of prime importance for the survival of an organism. Upon exposure to different damaging agents, DNA acquires various lesions such as base modifications, single-strand breaks (SSBs), and double-strand breaks (DSBs). Organisms have evolved specific repair pathways in order to efficiently correct such DNA damages. Among various types of DNA damages, DSBs are the most serious when present inside cells. Unrepaired or misrepaired DSBs account for some of the genetic instabilities that lead to secondary chromosomal rearrangements, such as deletions, inversions, and translocations and consequently to cancer predisposition. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher organisms. Mitochondrial DNA (mtDNA) deletions identified in humans are flanked by short directly-repeated sequences, however, the mechanism by which these deletions arise are unknown. mtDNA deletions are associated with various types of mitochondrial disorders related to cancer, aging, diabetes, deafness, neurodegenerative disorders, sporadic and inherited diseases. Compared to nuclear DNA (nDNA), mtDNA is highly exposed to oxidative stress due to its proximity to the respiratory chain and the lack of protective histones. DSBs generated by reactive oxygen species, replication stalling or radiation represents a highly dangerous form of damage to both nDNA and mtDNA. However, the repair of DSBs in mitochondria and the proteins involved in this repair are still elusive. Animals deficient for any one of the known Classical-NHEJ factors are immunodeficient. However, DSB repair (DSBR) is not eliminated entirely in these animals suggesting evidence of alternative mechanism, ‘alternative NHEJ’ (A-NHEJ/A-EJ). Several lines of evidence also suggest that alternative and less well-defined backup NHEJ (B-NHEJ) pathways play an important role in physiological and pathological DSBR. We studied NHEJ in different tissue mitochondrial protein extracts using oligomeric DNA substrates which mimics various endogenous DSBs. Results showed A-EJ, as the predominant pathway in mitochondria. Interestingly, immunoprecipitation (IP) studies and specific inhibitor assays suggested, mitochondrial end joining (EJ) was dependent on A-EJ proteins and independent of C-NHEJ proteins. Further, colocalization studies showed A-EJ proteins localize into mitochondria in HeLa cells. More importantly knockdown experiments showed the involvement of DNA LIGASE III in mitochondrial A-EJ. These observations highlight the central role of A-EJ in maintenance of the mammalian mitochondrial genome. By using oligomeric DNA substrates mimicking various endogenous DSBs, NHEJ in different cancer cell lines were studied. We found that the efficiency of NHEJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of NHEJ proteins. Interestingly, cancer cells with lower levels of BCL2 possessed efficient NHEJ and vice versa. Removal of BCL2 by immunoprecipitation and protein fractionation using size exclusion column chromatography showed elevated levels of EJ. Most importantly, the overexpression of BCL2 in vivo or the addition of purified BCL2 in vitro led to the downregulation of NHEJ in cancer cells. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo using immunoprecipitation and immunofluorescence, respectively. Hence, NHEJ in cancer cells is negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute towards increased chromosomal abnormalities in cancer. In summary, our study showed that the efficiency of EJ in cancers could be regulated by the antiapoptotic protein BCL2. However, it may not affect the mechanistic aspect of EJ. BCL2 instead may interfere with EJ by sequestering KU and preventing it from binding to DNA ends. This may help in better understanding towards increased chromosomal abnormalities in cancer. Study of mitochondrial DSBR in mammalian system highlights the central role of microhomology-mediated A-EJ in the maintenance of the mammalian mitochondrial genome and this knowledge will helpful for the development of future therapeutic strategies against variety of mitochondria associated diseases.

My research focuses on understanding the importance of human mitochondrial Hsp70 (Grp75) chaperone machinery for the maintenance of protein quality control inside the mitochondrial matrix. The investigations carried out during this study have been addressed towards gaining better insights into the working of Grp75 chaperone folding machinery in association with its diverse set of co-chaperones residing in human mitochondria. Additionally, the research also focuses on explaining the various modes of Grp75 participation leading to multiple disease conditions. The thesis has been divided into the following sections as follows: Chapter I: An introduction to the mitochondrial import machinery and role of mitochondrial Hsp70 chaperone folding machinery for the maintenance of protein quality control: Mitochondrion is an essential organelle present in the eukaryotic cell and requires more than 1500 proteins for its proper functioning. Although, mitochondria harbour their own genome, it encodes for only 13 proteins in humans. The rest of the entire proteome is encoded by the nuclear genome and requires proper targeting of proteins to different compartments of mitochondria. Remarkably, mitochondrial matrix alone requires more than 60% of the proteome for its suitable functioning. Briefly, the mitochondrial matrix destined polypeptide passes through the outer membrane translocon; the ‘TOM’ complex and then enters the TIM23 translocon present in the inner membrane of mitochondria. The complete translocation of the polypeptide into the mitochondrial matrix side requires the assistance of mtHsp70 based motor system present on the matrix side which pulls the polypeptide into the matrix in an ATP-dependent manner and with the assistance of various co-chaperones. Subsequently, the unfolded polypeptide is to be folded back to its native state, which is ensured again by the mtHsp70 based chaperone folding machinery. Importantly, while 20% of mtHsp70 is involved in protein import, 80% of mtHsp70 is dedicated for protein folding. In addition to mtHsp70, the chaperone folding machinery consists of various soluble co-chaperones such as the J-proteins which stimulate the ATP hydrolysis rate of Hsp70. Furthermore, another co-chaperone termed as a nucleotide exchange factor ensures binding of fresh ATP molecule onto Hsp70 ensuring multiple rounds of folding cycles. To understand the relevance of mitochondrial Hsp70 chaperone folding machine in the maintenance of protein quality control, Chapter I of the thesis has been divided into multiple sections as follows: Briefly, the initial portion of Chapter I provide a glimpse of the translocon components present in mitochondria for targeting of proteins to outer membrane, inner membrane and inter-membrane space. Owing to the vast proteome size of the mitochondrial matrix, the following section describes the detailed mechanism and translocation process of the mitochondrial matrix targeted proteins. Additionally, subsequent sections of Chapter I provide a comprehensive description of each of the mtHsp70 chaperone folding components, which maintain the protein quality control in the matrix. The players that constitute the chaperone folding machines are mitochondrial Hsp70, J-proteins, nucleotide exchange factors and the newly discovered human escort protein. Essentially, the section provides information about the cellular distribution, structure and function of each of these players constituting the mtHsp70 chaperone folding machine. Loss of regulation between these players leads to defects in protein folding. Imbalance in protein homeostasis is one of the primary causes for mitochondrial dysfunction leading to various diseases. Importantly, recent literature has highlighted the involvement of mtHsp70 chaperone folding players in Parkinson’s disease (PD), Myelodysplastic syndrome (MDS) and cancer. In accordance, the last section of the Chapter I has been dedicated to describe the basic cell biology and proposed mechanisms for the above diseased states. Interestingly, in comparison to yeast and bacteria, the composition of mtHsp70 chaperone folding machinery in humans is unique and distinctly different. Owing to a lack of information about the functioning of human mitochondrial Hsp70 chaperone folding machinery and with an emphasis on understanding its role in various disease manifestations, the objectives that were laid for my PhD thesis are as follows: 1) Functional in vitro reconstitution of the human Grp75 chaperone folding machinery by purifying all the Grp75 chaperone folding machinery players namely; Grp75 (human mtHsp70), hTid-1L and hTid-1S (J-proteins), GrpEL1 (nucleotide exchange factor) and Human escort protein (Hep). 2) Dissection of the intrinsic biochemical defects associated with the variants of Grp75 reported in Parkinson’s disease (PD). 3) To understand the correlation between elevated levels of Grp75 and its contribution to malignancy. In conclusion, the current study has highlighted some of the key features of human Grp75 chaperone folding machinery and its regulation in the maintenance of human mitochondrial matrix protein quality control, failure of which leads to pathological conditions. Chapter II: Reconstitution of the human Grp75 chaperone folding machinery to understand the functional interplay between the multiple protein components: The mitochondrial Heat shock protein 70 (mtHsp70) machinery components are highly conserved among eukaryotes, including humans. However, the functional properties of human mtHsp70 machinery components have not been characterized among all eukaryotic families. To study the functional interactions, we have reconstituted the components of mtHsp70 chaperone machine (Hsp70/J-protein/GrpE/Hep) and systematically analyzed in vitro conditions for biochemical functions. We observed that the sequence-specific interaction of human mtHsp70 towards mitochondrial client proteins differs significantly from its yeast counterpart Ssc1. Interestingly, the helical lid of human mtHsp70 was found dispensable to the binding of P5-peptide as compared to the other Hsp70’s. We observed that the two human mitochondrial matrix J-protein splice-variants differentially regulate the mtHsp70 chaperone cycle. Strikingly, our results demonstrated that human Hep possesses a unique ability to stimulate the ATPase activity of mtHsp70 as well as to prevent the aggregation of unfolded client proteins similar to J-proteins. We observed that Hep binds with the C-terminus of mtHsp70 in a full-length context, and this interaction is distinctly different from unfolded client-specific or J-protein binding. In addition, we found that the interaction of Hep at the C-terminus of mtHsp70 is regulated by the helical lid region. However, the interaction of Hep at the ATPase domain of the human mtHsp70 is mutually exclusive with J-proteins, thereby promoting a similar conformational change that leads to ATPase stimulation. Moreover, we have also dissected out the inter-domain defective nature associated with the point mutant of Grp75 implicated in Myelodysplastic syndrome thus providing an explanation for the loss of function of Grp75 eventually leading to loss of protein quality control in the diseased state. Chapter III: Enhanced J-protein interaction and compromised protein stability of Grp75 variants leads to mitochondrial dysfunction in Parkinson’s disease: Parkinson’s disease (PD) is the second most prevalent progressive neurological disorder commonly associated with impaired mitochondrial function in dopaminergic neurons. Although familial PD is multi-factorial in nature, a recent proteomic screen involving PD-patients revealed two mitochondrial Hsp70 variants (P509S and R126W) that are implicated in PD-pathogenesis. However, molecular mechanisms underlying how mtHsp70 PD-variants are centrally involved in PD-progression is totally elusive. In this report, we provide mechanistic insights into the mitochondrial dysfunction associated with human mtHsp70 PD-variants. Biochemically, R126W variant showed severely compromised protein stability and was found highly susceptible to aggregation at physiological conditions. Strikingly, on the other hand, P509S variant exhibits significantly enhanced interaction with J-protein co-chaperones involved in folding and import machinery, thus altering the overall regulation of chaperone mediated folding cycle and protein homeostasis. To assess the impact of mtHsp70 PD-mutations at the cellular level, we have developed yeast as a model system by making analogous mutations in Ssc1 ortholog. Interestingly, PD-mutations in yeast (R103W and P486S) exhibit multiple in vivo phenotypes, which are associated with ‘mitochondrial dysfunction’ such as mitochondrial DNA (mtDNA) loss and increased susceptibility to oxidative stress recapitulating the cellular features of dopaminergic neurons similar to those reported in other PD-models. Together, our observations for both the variants strongly indicate a definite involvement of mtHsp70 as a susceptibility factor in Parkinson’s disease. Chapter IV: To understand the correlation between elevated levels of Grp75 and its contribution to malignancy: Multiple studies carried out by various groups have reported the presence of elevated levels of Grp75 in cancer cells. Furthermore, proteomic screens show a positive correlation with the higher levels of Grp75 and the aggressive or metastatic nature of cancer. Importantly, cancer cells also exhibit altered mitochondrial metabolism and are found to be under constant oxidative stress pressure. Moreover, Grp75 actively participates in maintenance of mitochondrial function and as well is reported to interact with many putative oncoproteins. However, there is little information available on the possible role of Grp75 in modulating the cellular niche which might favor towards increased malignant transformation of cells. To identify pathways for explaining the correlation between Grp75 and cancer, our initial attempts have focused on monitoring the multiple cellular changes influenced by elevated levels of Grp75 in a cell line based system. To our surprise, transient transfection of cells with Grp75 led to a tremendous increase in the reactive oxygen species levels. Furthermore, a strong positive correlation between the extent of increased levels of Grp75 and the amount of ROS generated in these cells was established. As expected, increased ROS levels observed in Grp75 overexpressing cells also resulted in reduced cell viability. Notably, mitochondrial superoxide generation was found to be the major source for the observed increment in ROS levels in Grp75 expressing cells. In addition, the localization profile of the exogenously expressed Grp75 protein highlighted the fact that the protein was found to be predominantly targeted to mitochondria. Strikingly, the elevated Grp75 levels led to an increase in mitochondrial mass and also displayed a higher proportion of circular and fragmented mitochondria in these cells. Together, the above preliminary observations hint towards a strong correlation between the levels of Grp75 and its influence on the redox biology of cells providing an additional and a possible explanation of the mode of participation of Grp75 in generation and progression of malignancy.