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Autophagy is an evolutionarily conserved intracellular degradation process in which cytoplasmic components are captured in double membrane vesicles called autophagosomes and delivered to lysosomes for degradation. This process has an indispensable role in maintaining cellular homeostasis. The rate at which the dynamic turnover of cellular components takes place via the process of autophagy is called autophagic flux. In this review, we discuss about the orchestrated events in the autophagy process, transcriptional regulation, role of autophagy in some major human diseases like cancer, neurodegeneration (aggrephagy), and pathogenesis (xenophagy). In addition, autophagy has non-canonical roles in protein secretion, thus demonstrating the multifaceted role of autophagy in intracellular processes.

Unconventional protein secretion (UPS) is an important phenomenon with fundamental implications to cargo export. How eukaryotic proteins transported by UPS are recognized without a conventional signal peptide has been an open question. It was recently observed that a diacidic amino acid motif (ASP-GLU or DE) is necessary for the secretion of superoxide dismutase 1 (SOD1) from yeast under nutrient starvation. Taking cue from this discovery, we explore the hypothesis of whether the diacidic motif DE, which can occur fairly ubiquitously, along with its context, can be a generic signal for unconventional secretion of proteins. Four different contexts were evaluated: a physical context encompassing the structural order and charge signature in the neighbourhood of DE, two signalling contexts reflecting the presence of either a phosphorylatable amino acid (‘X’ in XDE, DXE, DEX) or an LC3 interacting region (LIR) which can trigger autophagy and a co-evolutionary constraint relative to other amino acids in the protein interpreted by examining sequences across different species. Among the 100 proteins we curated from different physiological or pathological conditions, we observe a pattern in the unconventional secretion of heat shock proteins in the cancer secretome, where DE in an ordered structural region has higher odds of being a UPS signal.

The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species—Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast., Author Summary Centromeres aid in high fidelity chromosome segregation. Paradoxically, centromere DNA sequences are rapidly evolving in fungi, plants, and animals. Centromere DNA sequences in fungi can be unique in each chromosome or share conserved features such as motifs for sequence specific protein binding, pericentric repeats, or transposon-rich elements. Ascomycetous fungi, in particular, show a wide range of diversity in centromere sequence elements. However, no ascomycetous budding yeast species is known to possess repeat-associated centromeres in all of its chromosomes. Here, we identified and mapped all seven centromeres in an ascomycete, a rapidly emerging human pathogenic yeast, Candida tropicalis. The repeat-associated centromeres of highly homogeneous DNA sequences in C. tropicalis are significantly diverged from the mostly non-repetitive unique centromeric DNA sequences of its closely related sequenced species, Candida albicans, Candida dubliniensis and Candida lusitaniae. Structurally, the centromeres of C. tropicalis more closely resemble those of the distantly related fission yeast Schizosaccharomyces pombe. Thus, we discover rapidly diverging repeat-associated centromeres in an ascomycetous budding yeast and provide evidence of emergence of repeat-associated centromeres via two independent evolutionary events in ascomycetous fungi.

We propose and demonstrate a light-sheet-based 3D interrogation system on a microfluidic platform for screening biological specimens during flow. To achieve this, a diffraction-limited light-sheet (with a large field-of-view) is employed to optically section the specimens flowing through the microfluidic channel. This necessitates optimization of the parameters for the illumination sub-system (illumination intensity, light-sheet width, and thickness), microfluidic specimen platform (channel-width and flow-rate), and detection sub-system (camera exposure time and frame rate). Once optimized, these parameters facilitate cross-scctional imaging and 3D reconstruction of biological specimens. The proposed integrated light-sheet imaging and flow-based enquiry (iLIFE) imaging technique enables single-shot sectional imaging of a range of specimens of varying dimensions, ranging from a single cell (HeLa cell) to a multicellular organism (C. elegans). 3D reconstruction of the entire C. elegans is achieved in real-time and with an exposure time of few hundred micro-seconds. A maximum likelihood technique is developed and optimized for the iLIFE imaging system. We observed an intracellular resolution for mitochondria-labeled HeLa cells, which demonstrates the dynamic resolution of the iLIFE system. The proposed technique is a step towards achieving flow-based 3D imaging. We expect potential applications in diverse fields such as structural biology and biophysics. Published by AIP Publishing.