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Rusan, N. M.

Publications and source records attributed to Rusan, N. M..

15 recordsLinked to original sources

Autobehaver: An AI-Based Pipeline for Animal Behavior Analysis

Behavior arises from the complex interplay between an organisms nervous system, its genetic makeup, and the environment. High-resolution, high-throughput behavioral quantification is essential for dissecting biological function and the effects of genetic perturbation, but automated analysis remains challenging. Here, we present Autobehaver, an automated behavioral analysis pipeline based on a low-cost, high-throughput recording platform that captures videos of individual Drosophila. From each video, we extracted keypoints and used a custom Transformer to assign frame-wise behavior and orientation labels. We then converted these predictions into high-dimensional per-animal feature vectors and trained XGBoost ensembles to classify animals and identify the features that separated groups. By applying SHAP analysis to the classifier ensemble, we identified the behavioral features most informative for distinguishing groups of flies. We demonstrated the approach in several ways. First, we recovered known behavioral changes associated with heat-activated dTrpA1 activity in specific neural circuits. Second, we detected age-associated behavioral changes consistent with gradual impairment of locomotor and climbing ability. Finally, we used Autobehavers classifier ensemble to place animals with intermediate phenotypes along a behavioral axis and used feature-importance analysis to reveal the behavioral features underlying those intermediate states. Together, Autobehaver provides an interpretable framework for quantitative behavioral phenotyping and comparative analysis of complex genotypes.

animal behavior and cognition↗

A MOPD II-associated Pericentrin variant disrupts PACT domain dimerization and pericentriolar material recruitment

Centrosome dysfunction is linked to developmental disorders affecting brain and body size, including microcephaly and primordial dwarfism. However, the cellular mechanisms underlying these rare conditions remain poorly understood. In this study, we investigate a rare variant of the centrosome-associated protein Pericentrin, which was discovered in a single family with Majewski/microcephalic osteodysplastic primordial dwarfism type II (MOPD II). Unlike the majority of pathogenic PCNT variants that cause severe protein truncation, the p.Lys3154del variant ({Delta}K3154) involves a single amino acid deletion in the proteins only conserved functional domain, providing a unique opportunity to explore PCNT function in MOPD II. To model PCNT{Delta}K3154, we examined the effects of Drosophila Pericentrin-like protein (PLP) carrying an orthologous deletion (Plp{Delta}R). Our results show that plp{Delta}R animals exhibit smaller tissues that recapitulate MOPD II phenotypes. Behavioral assays revealed defects in climbing and mechanosensation, suggesting impaired sensory cilia function. We also found that Plp{Delta}R cells exhibit accelerated mitosis, increased apoptosis, and reduced pericentriolar material recruitment. In silico structural modeling, yeast two-hybrid, and co-immunoprecipitation experiments show that Plp{Delta}R produces a protein that disrupts PLP dimerization and PLP interaction with Asterless, another centrosome protein. Overall, modeling the human MOPD II patient variant PCNT{Delta}K3154 in Drosophila reveals how a single amino acid deletion affects biological processes from the molecular level to the organismal level. Our work offers new insights into the defective cellular mechanisms underlying MOPD II in patients with the PCNT{Delta}K3154 variant, potentially linking the etiology of the disease in these individuals to the loss of a single protein-protein interaction.

cell biology↗

The Nuclear Pore Complex Facilitates Centriole-Nuclear Attachment in Spermatids

Proper connection between the sperm head and tail is critical for fertility and is mediated by the head-tail coupling apparatus (HTCA). Recent evidence suggests that the nuclear pore complex (NPC) may be important in male fertility, though a specific role at the HTCA has not been described. To investigate this, we performed a testis-specific RNAi screen targeting nucleoporins of the NPC. We identified Nup133 and Nup107 as regulators of HTCA development. We found that Nup133 and Nup107 were required to form the initial connection between the nucleus and centriole during HTCA establishment. We determined that failure to build the HTCA following Nup133 and Nup107 depletion was due to loss of nuclear envelope dynein/dynactin. Finally, we showed that loss of the NPC cytoplasmic filament component Nup358 results in the most severe centriole detachment phenotype, thus potentially functioning as the dynein anchor. Together, our data indicate that NPCs are critical regulators of early HTCA establishment and are required to recruit dynein to the nuclear envelope to bring the nucleus and centriole together during spermiogenesis.

cell biology↗

Togaram Ensures Axial Alignment of the Sperm Neck

The sperm neck must remain mechanically stable to preserve a straight head-tail axis during spermiogenesis, yet the mechanisms that maintain this alignment after head-tail attachment forms are poorly understood. Here, using Drosophila, we identify Togaram (Toga), the ortholog of mammalian TOGARAM1, as a regulator of this maintenance phase. Toga localizes to the sperm neck and to a broader perinuclear microtubule scaffold that surrounds the nucleus, which we term the Sperm Microtubule Cage (SMC). Depletion of Toga causes a striking off-axis phenotype in which the centriole usually remains attached to the nucleus but fails to maintain a straight head-tail axis, whereas complete detachment is comparatively rare. Live imaging shows that this defect arises after initial attachment has formed, indicating a maintenance defect rather than a failure of head-tail assembly. Mechanistically, Toga is dispensable for initial formation of head and neck microtubules but is required for their stabilization during Canoe-stage remodeling. Loss of Toga increases microtubule turnover, strongly reduces acetylated tubulin in the manchette, SMC, and neck region, and leaves the sperm neck vulnerable to buckling. Off-axis spermatids are depleted at later stages, consistent with selective loss of mechanically compromised cells. Finally, Cep104 and CCDC66 associate with Toga in cultured cells and are likewise required for axial alignment, consistent with a conserved module analogous to the mammalian ciliary tip module. Together, these findings support a model in which Toga stabilizes sperm head and neck microtubules to preserve a straight head-tail axis during the maintenance phase of spermiogenesis.

cell biology↗

The Proximal Centriole-Like Structure Anchors the Centriole to the Sperm Nucleus

Proper connection between the sperm head and tail is critical for sperm motility and fertilization. The link between the head and tail is mediated by the Head-Tail Coupling Apparatus (HTCA), which secures the axoneme (tail) to the nucleus (head). However, the molecular architecture of the HTCA is not well understood. Here, we use Drosophila to create a high-resolution map of proteins and structures at the HTCA throughout spermiogenesis. Using structured illumination microscopy, we demonstrate that key HTCA proteins Spag4 and Yuri form a Centriole Cap that surrounds the centriole (or Basal Body) as it is inserted, or embedded into the surface of the nucleus. As development progresses, the centriole is laterally displaces to the side of the nucleus, during which time the HTCA expands under the nucleus, forming what we term the Nuclear Shelf. We next show that the proximal centriole-like (PCL) structure is positioned under the Nuclear Shelf and functions as a critical stabilizer of the centriole-nuclear attachment. Together, our data indicate that the HTCA is complex, multi-point attachment site that simultaneously engages the PCL, the centriole, and the nucleus to ensure proper head-tail connection during late-stage spermiogenesis.

developmental biology↗

The UBR box E3 ligases Poe and Hyd are required for efficient Pericentrin degradation

Centrosomes are the major microtubule organizing centers (MTOC) of many cells and are composed of centrioles and the pericentriolar material (PCM). Centrosomes are an essential part of diverse cellular processes and require precise regulation of their protein levels. One protein whose levels must be regulated is Pericentrin - PCNT in humans and PLP in Drosophila. Increased PCNT expression and its protein accumulation is linked to clinical conditions including cancer, mental disorders, and ciliopathies. However, the mechanisms by which PCNT levels are regulated remains underexplored. Our previous study (Galletta, 2020) demonstrated that PLP levels are sharply downregulated during early spermatogenesis and this regulation is essential to spatially position PLP on the proximal end of centrioles. We hypothesized that the sharp drop in PLP protein was a result of a rapid protein degradation during the male pre-meiotic G2 phase. In this study we show that the N-terminal region of PLP is required for regulating its levels, which when elevated, extends PLPs position distally on centrioles. Consequently, PCM was also mispositioned leading to defects in spermatids. We then performed a targeted screen where we identified the E2 ligases, Rad6 and UbcD1, and the UBR box family E3 ligases, Poe (UBR4) and Hyd (UBR5) as factors that promote PLP degradation in spermatocytes. Collectively, our study suggests that Poe and Hyd directly bind the N-terminus of PLP and target it for degradation, ensuring proper centriole/basal body assembly and male fertility.

developmental biology↗

Pericentrin is a Kinesin-1 Activator that Drives Centriole Motility

Centrosome positioning is essential for their function. Typically, centrosomes are transported to various cellular locations through the interaction of centrosome nucleated microtubules with motor proteins. However, it remains unknown how centrioles migrate in cellular contexts in which centrioles do not nucleate microtubules. Here, we demonstrate that during interphase inactive centrioles move directly along the non-centrosomal microtubule network as cargo for the motor protein Kinesin-1. We identify Pericentrin-Like-Protein (PLP) as a novel Kinesin-1 interacting molecule essential for centriole motility. PLP directly interacts with the cargo binding domain of Kinesin-1 and they comigrate on microtubules in vitro. Finally, we demonstrate that PLP-Kinesin-1 dependent transport is essential for centrosome asymmetry age-dependent centrosome inheritance in asymmetric stem cell division.

cell biology↗

Spd-2 Gene Duplication Suggests Cell-Type Specific Assembly Mechanisms of Pericentriolar Material

Centrosomes are multi-protein complexes that function as the major microtubule organizing center (MTOC) for the cell. While centrosomes play tissue-specific MTOC functions, little is known about how particular centrosome proteins are regulated across cell types to achieve these different functions. To investigate this cell type-specific diversity, we searched for gene duplications of centrosome genes in the Drosophila lineage with the aim of identifying centrosome gene duplications where each copy evolved for specialized functions. Through in depth functional analysis of a Spd-2 gene duplication in the Willistoni group, we discovered differences in the regulation of PCM in somatic and male germline cells. The parental gene, Spd-2A, is expressed in somatic cells, where it can function to organize pericentriolar material (PCM) and the mitotic spindle in larval brain neuroblasts. Spd-2A is absent during male meiosis, and even when ectopically expressed in spermatocytes it fails to rescue PCM and spindle organization. In contrast, the new gene duplicate, Spd-2B, is expressed specifically in spermatocytes. During male meiosis, Spd-2B localizes to centrosomes, organizes PCM and spindles, and is sufficient for proper male fertility. Experiments using chimeric transgenes reveal that differences in the C-terminal tails of Spd-2A and Spd-2B are responsible for these functional changes. Thus, Spd-2A and Spd-2B have evolved complementary functions by specializing for distinct subsets of cells. Together, our results demonstrate that somatic cells and male germline cells have fundamentally different requirements for PCM, suggesting that PCM proteins such as Spd-2 is differentially regulated across cell types to satisfy distinct requirements. HighlightsO_LISpd-2 gene duplication in the Willistoni group is rapidly evolving C_LIO_LIParent gene Spd-2A is expressed in somatic cells, whereas the gene duplicate Spd-2B is expressed in spermatocytes C_LIO_LISpd-2A organizes pericentriolar material during somatic cell mitosis C_LIO_LISpd-2B is specialized for pericentriolar material organization during male meiosis C_LI

cell biology↗

Traip Mitotic Function Controls Brain Size

Microcephaly is a developmental failure to achieve proper brain size and neuron number. Mutations in diverse genes are linked to microcephaly, including several with DNA damage repair (DDR) functions; however, it is not well understood how these DDR gene mutations limit brain size. One such gene is TRAIP, which has multiple known functions in DDR. We characterized the Drosophila ortholog Traip, finding that loss of Traip causes a brain-specific defect in the Mushroom Body (MB). Traip mutant (traip-) MBs had reduced size and fewer neurons, but no neurodegeneration, consistent with human primary microcephaly disorders. Reduced neuron numbers in traip- were explained by premature caspase-dependent cell death of MB neuroblasts (MB-NBs). Many traip- MB-NBs had prominent chromosome bridges in anaphase, along with polyploidy, aneuploidy, or micronuclei. We found no evidence for an interphase DNA repair role for Traip in MB-NBs; instead, proper MB development requires Traip function during mitosis, where Traip localizes to centrosomes and mitotic spindles. Our results suggest that proper brain size is ensured by the recently described role for TRAIP in unloading stalled replication forks in mitosis, which suppresses DNA bridges and neural stem cell death to promote proper neuron number. Further, the mitotic nature of traip- MB-NB defects and Traip localization suggest a closer etiological link between DDR microcephaly genes like Traip and the centrosome/spindle-related genes more commonly associated with microcephaly.

developmental biology↗

Dynamic Sex Chromosome Expression in Drosophila Male Germ Cells

Sex chromosome gene content and expression is unusual. In many organisms the X and Y chromosomes are inactivated in spermatocytes, possibly as a defense mechanism against insertions into unpaired chromatin. In addition to current sex chromosomes, Drosophila has a small gene-poor X-chromosome relic (4th) that re-acquired autosomal status. Using single cell RNA-Seq, we demonstrate that the single X and pair of 4th chromosomes are specifically inactivated in primary spermatocytes. In contrast, genes on the single Y chromosome become maximally active in primary spermatocytes. Reduced X steady-state transcript levels are due to failed activation of RNA-Polymerase-II by phosphorylation of Serine 2 and 5. One Sentence SummarySex chromosome expression during spermatogenesis at the single cell level

developmental biology↗

A molecular mechanism for the procentriole recruitment of Ana2

Centriole duplication begins with the assembly of a pre-procentriole at a single site on a mother centriole and proceeds with the hierarchical recruitment of a conserved set of proteins, including Polo-like kinase 4 (Plk4)/ZYG-1, Ana2/SAS-5/STIL, and the cartwheel protein Sas6. During assembly, Ana2/STIL stimulates Plk4 kinase activity, and in turn, Ana2/STILs C-terminus is phosphorylated, allowing it to bind and recruit Sas6. The assembly steps immediately preceding Sas6-loading appear clear, but the mechanism underlying the upstream pre-procentriole recruitment of Ana2/STIL is not. In contrast to proposed models of Ana2/STIL recruitment, we recently showed that Drosophila Ana2 targets procentrioles independent of Plk4-binding. Instead, Ana2 recruitment requires Plk4 phosphorylation of Ana2s N-terminus, but the mechanism explaining this process is unknown. Here, we show that the amyloid-like domain of Sas4, a centriole surface protein, binds Plk4 and Ana2, and facilitates phosphorylation of Ana2s N-terminus which increases Ana2s affinity for Sas4. Consequently, Ana2 accumulates at the procentriole to induce daughter centriole assembly.

cell biology↗

Fascetto Interacting Protein (FIP) Regulates Fascetto (PRC1) to Ensure Proper Cytokinesis and Ploidy

Cell division is critical for development, organ growth, and tissue repair. The later stages of cell division include the formation of the microtubule (MT)-rich central spindle in anaphase, which is required to properly define the cell equator, guide the assembly of the acto-myosin contractile ring, and ultimately ensure complete separation and isolation of the two daughter cells via abscission. Much is known about the molecular machinery that forms the central spindle, including proteins needed to generate the antiparallel overlapping interzonal MTs. One critical protein that has garnered great attention is Protein Regulator of Cytokinesis 1 (PRC1), or Fascetto (Feo) in Drosophila, which forms a homodimer to crosslink interzonal MTs, ensuring proper central spindle formation and cytokinesis. Here, we report on a new direct protein interactor and regulator of Feo we named Fascetto Interacting Protein (FIP). Loss of FIP results in a significant reduction in Feo localization, rapid disassembly of interzonal MTs, and several cytokinesis defects. Simultaneous reduction in Feo and FIP results in tumor-like, DNA-filled masses in the brain. In aggregate our data show that FIP functions upstream of, and acts directly on, Feo to ensure fully accurate cell division.

cell biology↗

Micro computed tomography as an accessible imaging platform for exploring organism development and human disease modeling

Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering mechanisms driving animal development, physiology and disease. Elucidating these mechanisms has been enhanced through the study of model organisms and the use of sophisticated genetic, biochemical and imaging tools. Here we present an optimized method for non-invasive imaging of Drosophila melanogaster at high resolution using micro computed tomography (-CT). Our method allows for rapid processing of intact animals at any developmental stage, provides precise quantitative assessment of tissue size and morphology, and permits analysis of inter-organ relationships. We then use the power of -CT imaging to model human diseases through the characterization of microcephaly in the fly. Our work demonstrates that -CT is a versatile and accessible tool that complements standard imaging techniques, capable of uncovering novel biological mechanisms that have remained undocumented due to limitations of current methods.

developmental biology↗

An ordered pattern of Ana2 phosphorylation by Plk4 is required for centriole assembly

Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event, involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 binding stimulates Plk4 kinase activity in vitro, and, in turn, is phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STAN domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N-terminus of Ana2 which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4-Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.

cell biology↗

The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles

The centrosome serves as the main microtubule-organizing center in metazoan cells, yet despite its functional importance, little is known mechanistically about the structure and organizational principles that dictate protein organization in the centrosome. In particular, the protein-protein interactions that allow for the massive structural transition between the tightly organized interphase centrosome and the highly expanded matrix-like arrangement of the mitotic centrosome have been largely uncharacterized. Among the proteins that undergo a major transition is the Drosophila melanogaster protein centrosomin that contains a conserved carboxyl terminus motif, CM2. Recent crystal structures have shown this motif to be dimeric and capable of forming an intramolecular interaction with a central region of centrosomin. Here we use a combination of in-cell microscopy and in vitro oligomer assessment to show that dimerization is not necessary for CM2 recruitment to the centrosome and that CM2 alone undergoes a significant cell cycle dependent rearrangement. We use NMR binding assays to confirm this intramolecular interaction and show that residues involved in solution interactions are consistent with the published crystal structure and identify L1137 as critical for binding. Additionally, we show for the first time an in vitro interaction of CM2 with the Drosophila pericentrin-like-protein that exploits the same set of residues as the intramolecular interaction. Furthermore, NMR experiments reveal a calcium sensitive interaction between CM2 and calmodulin. Although unexpected because of sequence divergence, this suggests that centrosomin-mediated assemblies, like the mammalian pericentrin, may be calcium regulated. From these results we suggest a model where during interphase CM2 interacts with pericentrin-like-protein to form a layer of centrosomin around the centriole wall and that at the onset of mitosis this population acts as a nucleation site of intramolecular centrosomin interactions that support the expansion into the metaphase matrix.

biochemistry↗