Biology of the Cell

To increase our understanding of the inner working of cells, there is a need for specific markers to identify biomolecules, cellular structures and compartments. One type of markers comprises genetically encoded fluorescent probes that are linked with protein domains, peptides and/or signal sequences. These markers are encoded on a plasmid and they allow straightforward, convenient labeling of cultured mammalian cells by introducing the plasmid into the cells. Ideally, the fluorescent marker combines favorable spectroscopic properties (brightness, photostability) with specific labeling of the structure or compartment of interest. Here, we report our ongoing efforts to generate robust and bright genetically encoded fluorescent markers for highlighting structures and compartments in living cells. The plasmids are distributed by addgene: https://www.addgene.org/browse/article/28189953/ O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/160374v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@67f3aaorg.highwire.dtl.DTLVardef@ae2235org.highwire.dtl.DTLVardef@47f729org.highwire.dtl.DTLVardef@b3c9d1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Trypanosomatid parasites are kinetoplastid protists that compartmentalize glycolytic enzymes in unique peroxisome-related organelles called glycosomes. The heterohexameric AAA-ATPase complex of PEX1-PEX6 is anchored to the peroxisomal membrane and functions in the export of matrix protein import receptor PEX5 from the peroxisomal membrane. Defects in PEX1, PEX6 or their membrane anchor causes dysfunction of peroxisomal matrix protein import cycle. In this study, we identified the Trypanosoma PEX1 orthologue using sequence and structural similarities. Using yeast two-hybrid analysis, we demonstrate that TbPEX1 can bind to TbPEX6. Endogenously tagged TbPEX1 localizes to glycosomes in the T. brucei parasites. Depletion of PEX1 gene expression by RNA interference causes lethality to bloodstream form trypanosomes, due to a partial mislocalization of glycosomal enzymes to the cytosol and ATP depletion. TbPEX1 RNAi leads to a selective proteasomal degradation of both matrix protein import receptors TbPEX5 and TbPEX7. Unlike in yeast, PEX1 depletion did not result in an accumulation of ubiquitinated TbPEX5 in trypanosomes. As PEX1 turned out to be essential for trypanosomatid parasites, it could provide a suitable drug target for parasitic diseases. The results also suggests that these parasites possess a highly efficient quality control mechanisms that export the import receptors from glycosomes to the cytosol, in the absence of a functional TbPEX1-TbPEX6 complex.

FRET is a powerful tool to simultaneously establish and localize interactions between fluorescently tagged proteins with high spatial resolution. Rainey K.H. and Patterson G.H. introduced Photoswitching FRET (psFRET) using the Dronpa Photoswitching fluorescent protein. We present a straightforward detailed method, and a powerful software tool that allows adaptation of psFRET to diverse experimental setups. Image stacks, recording the decay of the Dronpa donor, serve as input to the software utility that includes effective preprocessing options preceding the calculation FRET efficiency at the single pixel level. We applied psFRET to generate interaction maps analyzing diverse interactions between cargo proteins, the GTPase Rab1b, and GRASP65 during ER to Golgi trafficking. Cargo-Rab1b interactions were restricted to the transit period from ER to Golgi. These data lend support to a mechanism whereby cargo sensing may regulate the level of downstream effectors recruitment to secretory membranes by Rab1.

An independent evaluation of the spectroscopic properties, cellular performance, merits and pitfalls of the redfluorescent protein mScarlet3-H as compared to mScarlet3 is reported. mScarlet3-H was generated from mScarlet3 by a single M163H mutation. Purified mScarlet3-H is characterized by a molar coeWicient of 79,040 M-1cm-1, afluorescence quantum yield of 17.8%, molecular brightness of 14.1 and a heterogeneous multiexponential decay with an averagefluorescence lifetime of 1 ns. Evaluation in living mammalian cells revealed a comparable maturation speed and eWiciency of mScarlet3 and mScarlet3-H, but the overall cellular brightness of mScarlet3-H was 5-fold lower than that of mScarlet3. Photobleaching analysis in live cells revealed identical photobleaching kinetics of mScarlet3-H and mScarlet-H. Thefluorescence intensity,fluorescence spectra andfluorescence lifetime of mScarlet3-H were found to be strongly pH-dependent between pH 4-8. Thefluorescence lifetime increased from 1 ns to 3 ns in lowering the pH from 8 to 4 with a pK of [~]6. The much lower lifetime of mScarlet3-H ([~]1 ns) as compared to mScarlet3 ([~]4 ns) allows dualfluorescence lifetime unmixing applications in single channel FLIM recordings in compartments with neutral to slightly alkaline pH. Furthermore, the strongly pH-dependentfluorescence lifetime of mScarlet3-H enablesfluorescence lifetime-based pH sensing in a pH region between pH 5 to 7. With this property autophagy of the cytoplasm can be visualized by the pH-dependentfluorescence lifetime with mScarlet3-H accumulation in lysosomes. Potential useful applications and pitfalls regarding the special properties of mScarlet3-H are discussed.

In this report, we describe the architecture of Lipofectamine 2000 and 3000 transfection- reagents, as they appear inside of transfected cells, using classical transmission electron microscopy (EM). We also demonstrate that they provoke consistent structural changes after they have entered cells, changes that not only provide new insights into the mechanism of action of these particular transfection-reagents, but also provide a convenient and robust method for identifying by EM which cells in any culture have been successfully transfected. This also provides clues to the mechanism(s) of their toxic effects, when they are applied in excess. We demonstrate that after being bulk-endocytosed by cells, the cationic spheroids of Lipofectamine remain intact throughout the entire time of culturing, but escape from their endosomes and penetrate directly into the cytoplasm of the cell. In so doing, they provoke a stereotypical recruitment and rearrangement of endoplasmic reticulum (ER), and they ultimately end up escaping into the cytoplasm and forming unique inclusion-bodies. Once free in the cytoplasm, they also invariably develop dense and uniform coatings of cytoplasmic ribosomes on their surfaces, and finally, they become surrounded by annulate lamellae of the ER. In the end, these annulate-lamellar enclosures become the ultrastructural signatures of these inclusion-bodies, and serve to positively and definitively identify all cells that have been effectively transfected. Importantly, these new EM-observations define several new and unique properties of these classical Lipofectamines, and allow them to be discriminated from other lipoidal or particulate transfection-reagents, which we find do not physically break out of endosomes or end up in inclusion bodies, and in fact, provoke absolutely none of these signature cytoplasmic reactions.

Trypanosoma brucei is a single celled eukaryotic parasite and the causative agent of human African sleeping sickness and Nagana in cattle. Aside from its medical relevance T. brucei has also been key to the discovery of several general biological principles including GPI-anchoring, RNA-editing and trans-splicing. The parasite contains a single mitochondrial organelle with a singular genome. Recent studies have identified several molecular components of the mitochondrial genome segregation machinery (tripartite attachment complex, TAC), which connects the basal body of the flagellum to the mitochondrial DNA of T. brucei. The TAC component in closest proximity to the mitochondrial DNA is TAC102. Here we apply and compare three different approaches (proximity labeling, Immunoprecipitation and yeast two-hybrid) to identify novel interactors of TAC102 and subsequently verify their localisation. Furthermore, we establish the direct interaction of TAC102 and p166 in the unilateral filaments of the TAC. Subject areabiochemistry, molecular biology, cellular biology

Small Ras-like GTPases are conserved across eukaryotes and mediate many functions. Trypanosoma brucei encodes only three putative open reading frames belonging to Ras and Rho subfamilies, all highly divergent from other taxa. We investigated the most Ras-like gene product in T. brucei, TbRlp, evaluating essentiality, location and impact of blocking the GDP/GTP cycle. TbRlp is expressed in both life stages, with a clear fitness contribution in bloodstream forms in in vitro culture, without a specific block to the cell cycle or clear morphological defect. Overexpression of TbRlp and mutant forms results in moderate disruption of the cell cycle and altered proliferation indicating that GTP/GDP cycling and expression levels of these forms of TbRlp are important to proliferation. Epitope-tagged TbRlp is located predominantly on structures in the posterior of the cell, and suggesting the late endosomal system. Analyses also indicates a link between cell density and levels of TbRlp in the BSF, with a decrease in TbRlp protein but not RNA upon increased cell density. These data suggest that TbRlp is important in control of proliferation and opens possibilities for future research.

Fluorescent reporters cover a wide range of applications in both basic and applied research. Whether a study involves microscopic imaging to study (co)-localization of proteins, FRET, biosensing, or quantifying gene expression, fluorophores are attractive reporter candidates due to their relatively straightforward in vivo readout. For microbiological applications, a wide variety of fluorescent proteins with varying excitation and emission wavelengths, brightness levels, and maturation times are available. Careful consideration is required when selecting from this large suite of proteins, especially when choosing multiple fluorophores. This is further complicated in phototrophic organisms, which exhibit strong autofluorescence, especially towards the red part of the spectrum, effectively eliminating common candidates such as mCherry. In this study, the specific properties and performance of a selection of fluorescent proteins are systematically evaluated against the background of photosynthetic pigment-derived autofluorescence in the cyanobacterium Synechocystis sp. PCC 6803. Specific readouts of different combinations of fluorescent proteins are also analyzed using high-throughput methods, namely plate reader fluorescent scans and single-cell flow cytometry to quantify fluorescence. The ultimate goal is to assess each fluorescent protein with regard to: 1.) Its ability to be discerned from cyanobacterial autofluorescence. 2.) Its compatibility with other fluorophores in this context. 3.) Its overall suitability in cyanobacterial research. Several highly suitable fluorescent proteins for use in cyanobacteria are identified, including mTagBFP2, mNeonGreen and mScarlet-I and suitable combinations, covering nearly the whole spectrum of visible light. This study expands the knowledge and toolset for current and future researchers and uncovers a whole spectrum of possibilities for fluorescent protein selection in cyanobacterial cell biology.

Carboxypeptidase E (CPE) a key factor in the biosynthesis of most peptide hormones and neuropeptides, is predominantly expressed in endocrine tissues and the nervous system. This highly conserved enzyme cleaves the C-terminal basic residues of the peptide precursors to generate their bioactive form. CPE is a secreted protein; however, the Intracellular pathways leading to its secretion are still obscure. We combined live-cell microscopy and molecular analysis to examine the intracellular distribution and secretion dynamics of fluorescently tagged CPE. CPE was found to be a soluble luminal protein as it traffics from the ER via the Golgi apparatus to lysosomes. Moreover, CPE is efficiently secreted and reinternalized to lysosomes of neighboring cells. The C-terminal amphipathic helix of CPE is essential for its efficient targeting to, and secretion from lysosomes. Fluorescence resonance energy transfer demonstrated that CPE and its substrate neuropeptide Y (NPY) interact in the Golgi apparatus and Immunoprecipitation analysis demonstrated that both CPE and NPY are co-secreted. The implications of the well-defined CPE intra and extracellular routes are discussed.

Nucleocytoplasmic transport is essential in eukaryotes and mediated by importin (Imp) /{beta} receptors that recognize nuclear localization signals (NLSs) on cargo proteins. The SV40 large T-antigen NLS (SV40-NLS), studied extensively, binds importin with high affinity in the nanomolar range, while modified versions, the Bimax peptides, bind in the picomolar range. Bimax peptides impede nuclear import and reduce viability in yeast and human cells, highlighting the potential of NLS peptides as inhibitors of nuclear transport. In this study, we investigated the potential of the SV40-NLS to target Toxoplasma gondii importin (TgImp). Expression of the SV40-NLS fused to a GFP reporter led to cytotoxicity in T. gondii tachyzoites; this depended on the SV40-NLS sequence and its position within the protein. Over-expression of TgImp rescued parasites from the SV40-NLS-induced cytotoxicity, confirming that the mechanism of action involves disruption of nuclear import. Importantly, the same construct did not affect cell viability in mammalian cells, suggesting a selective vulnerability in importin -mediated nuclear transport in the parasite. The SV40-NLS peptide offers an advantage over small-molecule inhibitors by targeting large interaction surfaces of importin with high specificity, minimizing off-target effects. This study lays the groundwork for a novel peptide-based therapeutic strategy employing NLS motifs to selectively inhibit nuclear import in T. gondii.

Nuclear-encoded mitochondrial proteins are correctly translocated to their proper sub-mitochondrial destination using location specific mitochondrial targeting signals (MTSs) and via multi-protein import machineries (translocases) in the outer and inner mitochondrial membranes (TOM and TIMs, respectively). However, MTSs of multi-pass Tims are less defined. Here we report the characterization of the MTSs of Trypanosoma brucei Tim17 (TbTim17), an essential component of the most divergent TIM complex. TbTim17 possesses a characteristic secondary structure including four predicted transmembrane (TM) domains in the center with hydrophilic N- and C-termini. After examining mitochondrial localization of various deletion and site-directed mutants of TbTim17 in T. brucei using subcellular fractionation and confocal microscopy we located at least two internal signals, 1) within TM1 (31-50 AAs) and 2) TM4 + Loop 3 (120-136 AAs). Both signals are required for proper targeting and integration of TbTim17 in the membrane. Furthermore, a positively charged residue (K122) is critical for mitochondrial localization of TbTim17. This is the first report of characterizing the internal mitochondrial targeting signals (ITS) for a multipass inner membrane protein in a divergent eukaryote, like T. brucei. SummaryInternal targeting signals within the TM1, TM4 with Loop 3, and residue K122 are required collectively for import and integration of TbTim17 in the T. brucei mitochondrion. This information could be utilized to block parasite growth.

Epsilon-tubulins are unconventional isoforms of the tubulin family, found in only a few organisms so far. We identified a novel epsilon-tubulin in Leishmania major (Lme-tubulin) that exhibits significant sequence similarity and conservation of functional domains with its known counterparts. Lme-tubulin was found to be constitutively expressed in both the extracellular promastigote form of the parasite and the amastigotes residing within infected macrophages. For localization studies, we generated a Leishmania strain expressing mNeonGreen-tagged Lme-tubulin using CRISPR-Cas9-mediated endogenous protein tagging method. Imaging studies with this strain revealed Lme-tubulin to be localized near the kinetoplast and at the flagellar base, indicating a basal body localization. That Lme-tubulin is indeed localized at the basal body and not a part of the microtubular network was confirmed when its localization was found to remain unaltered upon treatment with nocodazole, a microtubule disruptor. This is the first experimental finding of an e-tubulin not only in the genus Leishmania but in the entire Trypanosomatidae family and is likely to incite further research to uncover the physiological role of this intriguing tubulin in this group of protozoan parasites. Summary StatementWe report for the first time an epsilon-tubulin in Leishmania parasite. This nocodazole-insensitive unconventional tubulin is expressed constitutively and found to be localized in the basal body.

The accumulation of misfolded proteins inside the cells has been considered to be an important contributor to the development of cigarette smoke-mediated diseases. Since endocytosis plays a crucial role in protein trafficking and clearance, impaired endocytosis may contribute to cigarette smoke-mediated protein accumulation. Therefore, the current study investigated the effects of cigarette smoke extract (CSE) on the endocytosis process in yeast Saccharomyces cerevisiae. The current study showed that treatment of cells with CSE caused reduced uptake of FM4-64 stain, indicating impaired endocytosis. Further analysis revealed that CSE treatment resulted in a defect in the recruitment of proteins involved in endocytosis. Also, aberrant actin morphology was found upon CSE treatment, which might interfere with vesicle budding from the membrane. Moreover, the current study showed that the PI4,5P2 level in the plasma membrane in CSE treated cells is reduced due to the failed translocation of MSS4 kinase to the membrane. This reduced PI4,5P2 results in aberrant actin morphology. Thus, the current study demonstrates that CSE treatment causes endocytosis defects and provides insight into this defective endocytosis.

The optical refractive index of cellular components is generally not a property considered amenable to manipulation in microscopy as this is an intrinsic physical property of materials. Here we show that by targeting cephalopod reflectin protein nanoparticles one can manipulate the optical refractive index of mammalian cellular compartments. We further demonstrate that refractive index alteration based contrast agents can be utilized for dark field microscopy and quantitative phase contrast holotomography. Additionally we have molecularly cloned novel reflectins with improved and novel optical properties.

Endosomal sorting complex required for transport (ESCRT-III) is a membrane remodeling complex involved in a large number of cellular processes. It appears to perform an essential function in eukaryotes, since to date no eukaryotic organism completely devoid of ESCRT-III has been found. Yet, yeast cells with a deletion of all eight known ESCRT-III genes are viable. We therefore searched for new, previously undiscovered ESCRT-III like proteins in yeast. HHPred uncovered several proteins with similarity to Snf7. The similarity was mostly restricted to the 1-2 hairpin region of Snf7. A conserved pattern of amino acids was detected in this region. The protein encoded by ORF YPL199c strikingly resembled Snf7 in its secondary structure. Since this protein could be the ninth member of the ESCRT-III family in yeast, we called it Nbr9 ("number nine"). Nbr9 is palmitoylated and localizes to the plasma membrane. In contrast to other palmitoylated proteins, it is not associated with lipid rafts. When NBR9 was deleted in the octuple ESCRT-III deletion background, the yeast cells were still viable. However, despite a number of experiments, we do not have evidence at present that Nbr9 is part of an alternative ESCRT-III complex.

The endolysosomal tethering complexes HOPS and CORVET play pivotal roles in the homo- and heterotypic fusion of early and late endosomes, respectively, and HOPS also mediates the fusion of lysosomes with incoming vesicles including late endosomes and autophagosomes. These heterohexameric complexes share their four core subunits that assemble with additional two, complex-specific subunits. These features and the similar structure of the complexes allow the formation of hybrid complexes, and the complex specific subunits may compete for binding to the core. In our study, we decided to gain insight into how human HOPS and CORVET complexes form. We found that the overexpression of CORVET-specific Vps8 or Tgfbrap1 decreased the amount of core proteins Vps11 and Vps18 that are assembled with HOPS-specific subunits Vps41 or Vps39, which suggests reduced amount of intracellular HOPS. In line with this, we observed that the level of lipidated LC3 protein was elevated in these cells and the autophagic cargo p62 showed accumulation in cells overexpressing Vps8, suggesting failure in autophagosome-lysosome fusion in line with loss of HOPS function. In contrast, overexpression of HOPS-specific Vps39 or Vps41 did not affect the levels of these autophagic markers. Finally, we found that a hybrid complex containing Vps39 and Vps8 could be formed in HEK293 cells.

In cells, mRNA can be associated with various proteins, forming ribonucleoprotein complexes (RNPs) which take part in spatiotemporal control of translation. In the Drosophila melanogaster developing egg chamber, a set of RNPs is transported from the nurse cells to the oocyte and targeted selectively to specific cellular locations. This mRNA sorting process leads to the final oocyte polarization pre-defining the body axes of the future embryo. However, how mRNA is encoded for selection and directed transport is mechanistically not well understood. A master mRNA involved in body axes formation is bicoid, which localizes anterolaterally and is essential for head and thorax definition of the embryo. A protein that was identified essential for bicoid anterior localization is Exuperantia (Exu). Here, we use a live imaging-based pulse-chase approach, which reveals selective transport dynamics of Exu from nurse cells to the oocyte during mid to late-stage oogenesis.

African trypanosomes are medically important parasites that cause Sleeping sickness in humans and nagana in animals. In addition to their pathogenic role, they have emerged as valuable model organisms for studying fundamental biological processes. Protein tagging is a powerful tool for investigating protein localization and function. In a previous study, we developed two plasmids for rapid and reproducible protein tagging in trypanosomes, which enabled the localisation of all proteins in the trypanosome cell. However, the limited selection of fluorescent protein tags and selectable markers restricted the flexibility of this approach. Here, we present an expanded set of >100 vectors that utilizes universal primer annealing sequences, enabling protein tagging with a range of fluorescent and biochemical tags using five different selection markers. We evaluated the suitability of various fluorescent proteins for live cell imaging and determined their brightness and stability under different fixation conditions. Finally, we determined the optimal fluorescent protein for a set of specific experimental conditions demonstrating the utility of this toolkit.

Membrane-protein interactions mediate cellular invasion by toxins, and are thought to involve organized plasma membrane lipid domains, often containing glycolipids, other sphingolipids, and/or cholesterol. Here, we characterize an isolated glycolipid-interacting domain of the tetanus toxin heavy chain (Hc) as a fluorescently labelled peptide, TeNT46, and describe its membrane dynamics and binding characteristics on artificial bilayers and cellular membranes. We show that this novel ganglioside-interacting probe TeNT46 retains the glycolipid binding preferences of the parent toxin, using imaging-SPR (iSPR) on a micro-patterned hybrid bilayer surface. On live cell membranes, using fluorescence correlation spectroscopic (FCS) diffusion measurements to compare TeNT46 to the well-studied GM1-binding toxin CTxB, we find that both probes display ordered domain-binding characteristics, but distinct cholesterol and sphingolipid dependencies. Strikingly, the contrasting lipid requirements of TeNT46 from those of CTxB in cells are predicted by their iSPR binding preferences on hybrid synthetic membranes. Based on the combined findings from iSPR and FCS, we propose a model for toxin-membrane interaction whereby a unique lipid constellation determines optimum binding for each probe independently of lateral confinement, which is more generally imposed by cholesterol. Our resulting understanding of the specific lipid requirements of these toxin targets and their dynamics in cell membranes could be important for the future design of preventive membrane-based nano-decoys and cell-delivery tools.

Invariant chain (Ii) is an important multifunctional player in the regulation of adaptive immune responses and is responsible for several cellular functions related to MHCI and MHCII antigen loading and antigen presentation. While regulating endosomal trafficking of MHCII and other proteins that bind to Ii, this molecule is able to influence the endosomal pathway delaying the maturation of endosomes to the late endosomal loading compartments. When expressed in cells Ii is found to increase endosomal size, but the mechanisms for this is not known. We used RNAi silencing to identify SNARE proteins controlling Ii induced increase of endosomal size and delay of the endosomal pathway. Ii was found to interact with the SNARE protein Vti1b. Vti1b localized at the contact sites of fusing Ii positive endosomes and a tailless Ii was able to relocate Vti1b to the plasma membrane. Furthermore, silencing Vti1b, abrogated the delay in endosomal maturation induced by Ii expression. In conclusion, Ii interacts with Vti1b and this interaction is fundamental for Ii-mediated alteration of the endosomal pathway. We propose that Ii, by interacting with SNAREs, in particular Vti1B in the biosynthetic pathway of antigen presenting cells, is able to assemble SNARE directed fusion partners in the early part of the endosomal pathway that lead to a slower endosomal maturation for efficient antigen processing and antigen loading.