Kaplan, Oktay I. et al. (2019) International Worm Meeting "Functional characterisation of Joubert Syndrome associated CEP-41 and ARMC-9."

Kilic, Atiye, Altunkaynak, Betul, Pir, Mustafa S., Pir, Betul, Cevik, Sebiha, Yenisert, Ferhan, Kaplan, Oktay I.

[International Worm Meeting, 2019]

Joubert syndrome is a rare genetic disorder with an occurrence of approximately 1/100 000 live births. Developmental defects in cerebellum and brainstem are observed in patients with JS, and JS is defined as a neurodevelopmental disorder. Up to now 35 different genes have been linked to JS, and the functions of many of these genes including CEP41 and ARMC9 remain uncharacterised. To elucidate the functions of Joubert Syndrome associated CEP-41 (F42G8.19) and ARMC-9 (F59G1.4) genes, we have generated transgenics containing their own promoter driven GFP labelled constructs and generated/obtained null mutants for these genes. CEP-41 was found to be exclusively expressed in the ciliated sensory neurons, and its localisation is restricted to the middle segment of cilia while ARMC-9 localizes to the entire length of the ciliary axonemes. While null mutants for these genes possess no abnormal cilia structures, a number of double mutant combinations of cep-41 with other ciliopathy genes reveal defects in cilia structure in C. elegans.

Kaplan, Oktay Ismail et al. (2013) International Worm Meeting "Conversion of epithelial cells into a neuron like cell in C. elegans."

Kaplan, Oktay Ismail, Tursun, Baris, Bulut, Idris Selman

[International Worm Meeting, 2013]

Cell reprogramming has been an intensive focus of research in recent years due to its great potential for new medical applications. Different procedures such as converting one cell type to another distinct cell type either through generating stem cell-like states in vitro or by direct reprogramming are continuously being explored. One limitation is that many cells display resistance to be directly reprogrammed probably due to the absence of "co-activators" or presence of "inhibitory factors" required for cell reprogramming. C. elegans has recently emerged as a genetic tool to identify "inhibitory factors" required for cell fate reprogramming. A recent reverse genetic analysis of chromatin factors has successfully revealed a role for the conserved gene lin-53 (mammalian Rbbp4/7) in reprogramming mitotic germ cells into neuron and muscle like-cells, depending on induction of fate-inducing transcription factors such as CHE-1 or HLH-1 (MyoD), respectively. However, forced reprogramming of differentiated somatic cells into neurons has remained to be difficult in C. elegans (Tursun et al. 2011, Patel et al. 2012). Towards uncovering novel inhibitory factors required for conversion of differentiated somatic cells into a neuronal like cell, we have conducted a forward genetic screen. Our screen has identified numerous mutant backgrounds, where ectopic expression of che-1 (induces glutamatergic ASE neuron fate) induces expression of numerous neuronal genes in epidermis, potentially overcoming a barrier for directly reprogramming epidermal cells into neuron-like cells. Our findings suggest that, in addition to germ cells, differentiated somatic cells in C. elegans have the potential to be reprogrammed into neuron like cells. We are in the process of mapping genes responsible for restricting the reprogramming of differentiated somatic cells. References: 1)Patel T, Tursun B et al Removal of Polycomb Repressive Complex 2 makes C. elegans germ cells susceptible to direct conversion into specific somatic cell types. Cell Reports 2012; in press (DOI: 10.1016/j.celrep.2012.09.020) 2)Tursun B, Patel T et al Direct conversion of C. elegans germ cells into specific neuron types Science 2011; 331: 304-308.

Kaplan, Oktay Ismail et al. (2015) International Worm Meeting "Uncovering Molecular Barriers to Direct Cell Reprogramming in C. elegans."

Kaplan, Oktay Ismail, Bulut, Idris Selman, Tursun, Baris

[International Worm Meeting, 2015]

Cells become progressively committed towards a specific identity during embryonic development. Techniques such as transdifferentiation by overexpressing of a cell fate-inducing factor or reprogramming to induced pluripotent stem cells (iPSC) proved that differentiated cell fates are not fixed but reversible. Cell fate conversion strategies provide a great promise for cell therapy. However, direct cell reprogramming by transdifferentiation, which can be performed in vivo in contrast to the iPSC procedure, proved to be difficult since most differentiated cells display resistance once terminal differentiation occurs. The molecular mechanisms preventing differentiated cells to be converted by a cell fate-inducing factor remain elusive. We devised a non-biased approach to recover genetic backgrounds that overcome the molecular barrier for cell reprogramming. Our screen identified 17 mutant backgrounds, where ectopic overexpression of the ASE neuron-inducing factor CHE-1 leads to induction of ASE-specific cell fate markers in germ cells and/or epidermal cells. For example, once che-1 mis-expression is broadly induced, a number of mutants including bar1, bar2, bar4, bar6 and bar13 start to express the ASE cell fate marker gcy-5::gfp in the epidermis. while bar1, bar9, bar10 and bar11 allow induction of ASE cell fate markers in germ cells. We have mapped the relative position of mutations and identified the responsible genes in a number of mutants using the SNP strategy paired with next-generation sequencing technologies. For example, bar1 and bar2 are alleles of usp-48 encoding a conserved ubiquitin specific peptidase and was proposed to act as a chromatin eraser for a number of histone marks (1). Presently, we are in the process of characterizing and mapping other bar mutants. Reference:1- X Ji, DB Dadon, BJ Abraham, TI Lee et al (2015),Chromatin proteomic profiling reveals novel proteins associated with histone-marked genomic regions, PNAS, 3841-3846, doi: 10.1073/pnas.1502971112.

Kaplan, Oktay et al. (2009) International Worm Meeting "Distinct roles for C. elegans clathrin adaptin complexes AP1 and AP2 in mediating protein trafficking to cilia."

Blacque, Oliver, Kaplan, Oktay, Kida, Katarzyna

[International Worm Meeting, 2009]

Clathrin in association with four adaptor complexes (AP1-4) facilitates membrane trafficking and protein sorting between various cellular compartments. Despite increasing evidence implicating these systems in ciliary processes, the role of metazoan clathrin and AP complexes in cilium formation and function remains poorly understood. Here we addressed this question in C. elegans sensory neurons. First, we found that clathrin heavy chain (CHC-1), AP1 (UNC-101, APS-1) and AP2 (DPY-23, APA-2) subunits, but not AP3 subunits, are required for normal cilia integrity, morphology and ultrastructure. Subcellular localisation analysis using rescuing GFP reporters revealed that AP1 and AP2 subunits display distinct non-ciliary localisations, with UNC-101 and APS-1 predominantly within the perinuclear region, whereas DPY-23 is found more diffusely throughout the cell, including a region near the ciliary base. Loss of CHC-1, AP1 and AP2 subunit function results in overlapping yet distinct defects in ciliary protein trafficking. In chc-1 and unc-101 mutants, ODR-10 (transmembrane protein) is trapped at the plasma membrane and fails to undergo dendritic translocation, whereas in dpy-23 mutants, ODR-10 translocates normally along dendrites but frequently accumulates within the distal dendrite region. Consistent with differential effects of AP1 and AP2 subunits on membrane trafficking to cilia, the assembly of enlarged AWB ciliary membrane fans in grk-2 mutants requires unc-101, but not dpy-23. Similarly, loss of CHC-1 and AP1/2 function differentially affects intraflagellar transport, with IFT grossly normal in unc-101 mutants, partially slowed in dpy-23 mutants and absent altogether in chc-1(RNAi) animals. Finally, loss of chc-1 and dpy-23 function, but not unc-101, results in the distal dendrite accumulation of IFT proteins. Together, these data demonstrate roles for metazoan CHC, AP1 and AP2, but not AP3, in cilium formation. In addition, these findings suggest that AP1 and AP2 operate at distinct ciliary protein sorting compartments, mediating differential effects on membrane trafficking to cilia and intraflagellar transport.

Oktay Ismail Kaplan et al. (2008) European Worm Meeting "Investigation of protein targeting to C. elegans sensory cilia"

Oktay Ismail Kaplan, Oliver Blacque

[European Worm Meeting, 2008]

Primary cilia are microtubule-based organelles found on the surface of. eukaryotic cells, where they play critical roles in chemosensation,. signalling and vertebrate development. Mutations in genes associated with. cilia biogenesis and function underlie several human disease phenotypes. including cystic kidneys, retinal degeneration and Bardet-Biedl syndrome. (BBS). Cilia are built and maintained by intraflagellar transport (IFT), a. bi-directional motor-driven motility of protein complexes along ciliary. axonemes that is thought to deliver various protein cargos (e.g.,. structural ciliary components, signalling molecules, membrane. receptors/channels) to the organelle. Although IFT likely serves as a. downstream distribution mechanism of targeting proteins to discrete parts. of ciliary structures, the upstream events of how proteins are targeted. from their site of synthesis (in the cell body) to the ciliary base region. is very poorly understood.. To investigate this question, we employ C. elegans, where neuronal. sensory cilia extend from the distal tip of long dendrite structures. In. this study, we are performing microscopy-based genetic screens (using EMS. mutagenesis) to identify mutations that disrupt trafficking of GFP-tagged. ODR-10 to AWB cilia. ODR-10 is a 7-transmembrane protein that functions as. a receptor to the volatile odorant diacetyl, and as shown previously, ODR-. 10::GFP localises very strongly in an unc-101-dependedent manner to wild-. type AWB and is observed to translocate along the dendritic compartment. [1]. So far in this study, approximately 1000 mutagenised genomes have been. screened, and 14 mutants recovered with defects in ODR-10::GFP ciliary. localisation. A number of these mutants are Dyf(+), indicating that the. inability of ODR-10 to localise to cilia is not due to a significant loss. of ciliary structure and may instead be due to defects in the targeting. mechanism.. 1. Dwyer ND, Adler CE, Crump JG, L''Etoile ND, Bargmann CI. Polarized. dendritic transport and the AP-1 mu1 clathrin adaptor UNC-101 localize. odorant receptors to olfactory cilia. Neuron 31, 277-87 (2001).

Cevik, Sebiha et al. (2021) MicroPubl Biol

Cevik, Sebiha, Kaplan, Oktay I.

[MicroPubl Biol, 2021]

We report the identification of C. elegans F42G8.19 as the ortholog of human CEP41. This conclusion was based on database searches, reciprocal BLAST analysis, and cilia-specific localization. To look for the orthologous gene of human CEP41 in C. elegans, we used three databases: the model organism Alliance of Genome Resources (https://www.alliancegenome.org/, Release 3.2.0), OrthoList 2 (http://ortholist.shaye-lab.org/, Release 2017), and ConVarT (https://convart.org/, Release 2020) (Kim et. al., 2018; Pir et. al., 2021). On the Alliance of Genome Resources and ConVarT, we discovered that C. elegans F42G8.19 is the ortholog of human CEP41. To validate this conclusion, we ran a manual reciprocal BLAST analysis (the Reciprocal Best Hits BLAST (RBHB)). The protein-protein BLAST (BLASTp) search of human CEP41 protein sequence (NP 061188.1) revealed F42G8.19 as the top C. elegans hit (Altschul et. al., 1990). In the following step, the protein sequence from the best hit C. elegans F42G8.19 (NP_001294206.1) was compared to human proteins, with human CEP41 emerging as the best match. C. elegans F42G8.19 encodes a 182-amino-acid protein that is shorter than the 373-amino-acid human CEP41 protein (Figure 1A). We subsequently performed the amino acid alignments of human CEP41 (169373 amino acid) and C. elegans F42G8.19 (1182 amino acid), which revealed over 24 % identity to each other (Figure 1A). We eventually determined whether C. elegans F42G8.19 has a rhodanese domain as human CEP41 (169-266 aa), and our query showed that C. elegans F42G8.19 has a rhodanese domain (30-121 aa) (Figure 1A) (Lee et. al., 2012). Taken together, our analysis revealed that C. elegans F42G8.19 is orthologous to human CEP41, and C. elegans F42G8.19 was therefore assigned CEPH-41 (CEntrosomal Protein Homolog 41).

Cevik, Sebiha et al. (2021) MicroPubl Biol

Cevik, Sebiha, Kaplan, Oktay I.

[MicroPubl Biol, 2021]

C. elegans EGL-36 displays extensive similarity to human KCNC proteins, the conserved potassium channel subfamily KV3 (the Shaw subfamily). This potassium channel subfamily contains four genes KCNC1, KCNC2, KCNC3, and KCNC4 encoding KV3.1 (Shaker), KV3.2 (Shab), KV3.3 (Shaw), and KV3.4 (Shal), respectively. Previous studies have demonstrated that C. elegans EGL-36 is expressed in the egg-laying muscles, ventral cord neurons, and the subset of ciliated sensory neurons including ADEL, ADER, PHB through its subcellular localization in the ciliated sensory neurons remains unknown (Elkes et al. 1997; Johnstone et al. 1997). To observe the subcellular localization of EGL-36 in the ciliated sensory neurons in C. elegans, we tagged green fluorescent protein (GFP) at the C-termini of EGL-36 with a cilia-specific promoter. We observed overexpressed EGL-36::GFP in the subsets of sensory neurons in the head and tail including PHB sensory neurons in the tail but not in the PHA sensory neuron, which is in line with previous findings (Elkes et al. 1997; Johnstone et al. 1997) (Figure 1A). Confocal laser scanning microscopy shows that EGL-36::GFP is predominantly located at the base of the cilium. The EGL-36::GFP signal can be found in the neuronal cell body, with weak dendrite staining, where they may function in the dendritic excitability (Figure 1A).

KONAC, Ahsen et al. (2021) International Worm Meeting "Understanding the Molecular Basis of Aging of Sensory Neurons in C. elegans"

KONAC, Ahsen, Cevik, Sebiha, Kaplan, Oktay

[International Worm Meeting, 2021]

Though aging is inevitable, an average human life expectancy has substantially increased for the last 150 years, but the extension of lifespan does not necessarily equate with improved healthspan. The nervous system and neuronal cells gradually deteriorate with aging, but the molecular basis of neuronal aging, especially the aging of sensory neurons and sensory functions, remains a mystery. We employ C. elegans to address the following questions: (1) When does aging in the sensory neurons and sensory neurons related functions start? (2) Does aging in the neurons always occur at the same time or do different neuronal cells display distinct patterns? (3) Can we slow down neuronal aging? To this end, we generated several transgenic strains with various neuronal markers (endogenous and fosmid-based) labeled with various fluorescent tags. Then, using our fluorescence-based system, we examined whether a variety of mutant strains could reverse the neuronal aging. Our initial analysis has started to answer some of these questions. We will present the initial results in the meeting.

Zorluer, Atiyye et al. (2021) International Worm Meeting "Use of C. elegans for investigating functional consequence of orthologous variants"

Kaplan, Oktay I., Zorluer, Atiyye, Cevik, Sebiha

[International Worm Meeting, 2021]

The widespread use of next-generation sequencing technologies has resulted in the emergence of millions of new genetic variants in humans and non-human species. One of the most important key tasks in genomics is to differentiate harmful genetic variants from non-harmful variants to diagnose disease better. Computational prediction tools and experimental data have helped to shed light on the functional importance of human genetic variants in this respect. However, mutants bearing human equivalent variants, known as orthologous variants (OrthoVar), have gotten less attention, even though they can be beneficial resources for unraveling the functional data for human OrthoVars. Here we used ConVarT (https://convart.org/), an OrthoVar search engine, to look for human OrthoVars in C. elegans. We selected ift-140, which encodes ciliopathy associated intraflagellar transport 140, because a null mutation causes easily detectable cilia abnormality in C. elegans. We identified conserved amino-acid positions that undergo amino acid changes within IFT-140 and discovered seven different variants that fall into this category, three of which have human OrthoVars. We obtained variant carrying mutants from CGC generated by the Million Mutation project and performed a fluorescent dye uptake assay, which indirectly assesses cilia structure. Except for V444I, none of these mutants exhibit Dye uptake defect, indicating that these variants are likely non-harmful variants. V444I mutants have a temperature-sensitive Dye uptake defect as well as IFT protein accumulations in the cilia, which is similar to the ift-140 null mutant phenotype. We are currently performing a rescue analysis for this variant. We then generated two mutants with human OrthoVars (G680S, P702A), and our thorough analysis found that the P702A variant (Human P726A, reported likely pathogenic) is a null mutant of ift-140, but not G680S (Human G704S, VUS). Our current OrthoVar-focused study has uncovered the functional implications of nine variants (5 human OrthoVars), and we believe that functional assessment of human OrthoVars can provide valuable insight into human variants.

Turan, Merve et al. (2021) International Worm Meeting "Molecular Mechanism of Coordinating Cilia Intersection and Elongation"

Turan, Merve, Cevik, Sebiha, Kaplan, Oktay

[International Worm Meeting, 2021]

Cilia and flagella are motile or non-motile cellular organelles that track their surroundings and/or coordinate cell/organism motility. Even though multiple motile cilia are bundled together on a cell surface, nonmotile cilium elongates from the cell surface as one cilium. Non-motile cilia from separate cells appear to form joint cilia by extending in parallel to each other. The non-motile cilia of C. elegans PHA and PHB sensory neurons, for example, protrude from the end of the dendrite but they extend side by side and intersect in the middle part of the cilia, reaching the same length. However, the molecular mechanisms underlying how adjacent cilia achieve the same length and converge remain a mystery. In the current study, we employ C. elegans to investigate the molecular mechanisms underlying these characteristics. We discovered that several genes, including arl-13, are needed for the correct convergence of PHA and PHB cilia. We next created a list of genes expressed in either of these two ciliated cells (PHA or PHB cells). For example, the conserved potassium voltage-gated channel gene egl-36 is expressed in PHB cells and not in PHA cells, and we found that it localizes at the base of the cilia. We generated egl-36 mutations in combination with other ciliary gene mutants because the disruption function of egl-36 alone does not cause gross ciliary defects. In these double mutants, we observed short cilia in both PHB and PHA sensory neurons. We are gathering more data for this study right now, and we will present our findings at the conference. Key words: cilia, cilia biogenesis, arl-13; egl-36