SPC25 Functions as a Prognostic-Related Biomarker, and Its High Expression Correlates with Tumor Immune Infiltration and UCEC Progression.

BACKGROUND: Most tumor tissues expressed spindle pole body component 25 (SPC25), one of the four subunits of the NDC80 complex, at greater levels compared to surrounding normal tissues. According to earlier researches, this subunit strongly encouraged tumor cell proliferation and tumor growth, which resulted in worse prognoses in patients with hepatocellular, breast, lung, and prostate cancer. Precisely because SPC25's role in uterine corpus endometrial carcinoma (UCEC) is understudied, we chose to concentrate on UCEC for gaining a more scientific and thorough understanding of SPC25. METHODS: Along with examining SPC25's differential expression, prognostic significance, and biological function in UCEC, our research sought to clarify the underlying mechanism by which SPC25 influences the course of UCEC and patient prognosis from the viewpoints of methylation and immune infiltration. RESULTS: We observed differential expression of SPC25 gene in different clinicopathological features of UCEC and identified SPC25 as a hazard factor for poorer overall survival (OS), disease-specific survival (DSS), and progress free interval (PFI) in UCEC, particularly in its multiple clinical subtypes. In addition, we also discovered that SPC25 and its co-expressed genes mostly engaged in biological processes and signal transduction routes linked to cell cycle and cell division in UCEC. After investigating SPC25's methylation status, we discovered that patients with UCEC had elevated SPC25 expression and a poor prognosis due to hypomethylation of CpG sites in the SPC25 gene sequence. Finally, we investigated SPC25's potential role in immunotherapy and discovered that SPC25 might alter the major immune cell infiltration levels in the tumor microenvironment (TME) by regulating the expression of immunoregulatory molecules and chemokines, which would be beneficial for SPC25 to control the progression of UCEC. CONCLUSIONS: In conclusion, SPC25 was a useful predictive biomarker as well as a possible therapeutic target for UCEC.

SPC25 as a novel therapeutic and prognostic biomarker and its association with glycolysis, ferroptosis and ceRNA in lung adenocarcinoma.

OBJECTIVE: Spindle pole body component 25 (SPC25) is an important cyclin involved in chromosome segregation and spindle dynamics regulation during mitosis. However, the role of SPC25 in lung adenocarcinoma (LAUD) is unclear. MATERIALS AND METHODS: The differential expression of SPC25 in tumor samples and normal samples was analyzed using TIMER, TCGA, GEO databases, and the correlation between its expression and clinicopathological features and prognosis in LUAD patients. Biological pathways that may be enriched by SPC25 were analyzed using GSEA. In vitro cell experiments were used to evaluate the effect of knocking down SPC25 expression on LUAD cells. Correlation analysis and differential analysis were used to assess the association of SPC25 expression with genes related to cell cycle, glycolysis, and ferroptosis. A ceRNA network involving SPC25 was constructed using multiple database analyses. RESULTS: SPC25 was highly expressed in LUAD, and its expression level could guide staging and predict prognosis. GSEA found that high expression of SPC25 involved multiple cell cycles and glycolytic pathways. Knocking down SPC25 expression significantly affected the proliferation, migration and apoptosis of LUAD cells. Abnormal SPC25 expression levels can affect cell cycle progression, glycolytic ability and ferroptosis regulation. A ceRNA network containing SPC25, SNHG15/hsa-miR-451a/SPC25, was successfully predicted and constructed. CONCLUSIONS: Our findings reveal the association of up-regulation of SPC25 in LUAD and its expression with clinical features, prognosis prediction, proliferation migration, cell cycle, glycolysis, ferroptosis, and ceRNA networks. Our results indicate that SPC25 can be used as a biomarker in LUAD therapy and a target for therapeutic intervention.

Activation of the DNA damage checkpoint perturbs asymmetric localization of Kar9 to spindle pole bodies in Saccharomyces cerevisiae.

During cell cycle progression in Saccharomyces cerevisiae, spindle pole bodies (SPBs) are duplicated during the G1/S-phase transition. SPBs are crucial for the organization of both the spindle and astral microtubules, and their orientation defines the direction of nuclear division. In this process, an old SPB, which serves as the template SPB during the duplication process, is oriented toward the bud side. The patterning microtubule plus-end tracking protein, Kar9, plays an important role in the orientation of SPBs by asymmetrically localizing to the old SPB. Here, methylglyoxal (MG), a metabolite derived from glycolysis, was found to perturb asymmetric Kar9 localization and influence proper positioning of the old SPB. MG activated the DNA damage checkpoint pathway, and MG-induced perturbation of asymmetric Kar9 localization was abolished by the deletion of MEC1, a sensor for the DNA damage checkpoint pathway. Methyl methanesulfonate, a DNA-alkylating agent, also perturbed asymmetric Kar9 localization. Our results suggest that activation of the DNA damage checkpoint pathway perturbs the asymmetric Kar9 localization required for proper positioning of SPBs.

Direct recruitment of Mis18 to interphase spindle pole bodies promotes CENP-A chromatin assembly.

CENP-A chromatin specifies mammalian centromere identity, and its chaperone HJURP replenishes CENP-A when recruited by the Mis18 complex (Mis18C) via M18BP1/KNL2 to CENP-C at kinetochores during interphase. However, the Mis18C recruitment mechanism remains unresolved in species lacking M18BP1, such as fission yeast. Fission yeast centromeres cluster at G2 spindle pole bodies (SPBs) when CENP-ACnp1 is replenished and where Mis18C also localizes. We show that SPBs play an unexpected role in concentrating Mis18C near centromeres through the recruitment of Mis18 by direct binding to the major SPB linker of nucleoskeleton and cytoskeleton (LINC) component Sad1. Mis18C recruitment by Sad1 is important for CENP-ACnp1 chromatin establishment and acts in parallel with a CENP-C-mediated Mis18C recruitment pathway to maintain centromeric CENP-ACnp1 but operates independently of Sad1-mediated centromere clustering. SPBs therefore provide a non-chromosomal scaffold for both Mis18C recruitment and centromere clustering during G2. This centromere-independent Mis18-SPB recruitment provides a mechanism that governs de novo CENP-ACnp1 chromatin assembly by the proximity of appropriate sequences to SPBs and highlights how nuclear spatial organization influences centromere identity.

Correlation Between Serum ESPL1 and Hepatitis B Virus-related Hepatocellular Carcinoma Histological Grade: A Chinese Single-center Case-control Study.

BACKGROUND/AIM: Serum markers to determine the histological grade of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) are still limited. This study aimed to investigate if serum extra spindle pole bodies-like 1 (ESPL1) protein could reflect the histological grade of HBV-related HCC. MATERIALS AND METHODS: A total of 154 patients with HBV-related HCC were enrolled in the experimental group and 41 non-HBV-related patients in the control. Enzyme-linked immunosorbent assay was used to detect serum ESPL1 levels. The differences in serological ESPL1, alpha-fetoprotein (AFP), and des-gamma-carboxy prothrombin (DCP) were compared between the two groups. HCC tumor diameter was measured, and pathological examination was performed to compare the relationship between ESPL1, AFP, and DCP and tumor size and histological grade. RESULTS: Serum AFP and DCP levels showed no significant difference between experimental group and control group, and increased when the tumor diameter increased but were not related to HCC histological grade. Serological ESPL1 levels were higher in the experimental group than those in the control group, and positively correlated with the histological grade. In the experimental group, tumor size and histological grade were almost independent (Kappa=0.000); patients with medium size tumors had the highest serum ESPL1 levels and the highest proportion of poorly differentiated carcinomas, whereas 75.6% of patients with small size tumors had moderately differentiated carcinomas and only 20% well differentiated carcinomas. CONCLUSION: Serum ESPL1 can reflect the malignant degree of HBV-related HCC and is helpful in identifying small size HCC tumors.

Characterization of a novel interaction of the Nup159 nucleoporin with asymmetrically localized spindle pole body proteins and its link with autophagy.

Both the spindle microtubule-organizing centers and the nuclear pore complexes (NPCs) are convoluted structures where many signaling pathways converge to coordinate key events during cell division. Interestingly, despite their distinct molecular conformation and overall functions, these structures share common components and collaborate in the regulation of essential processes. We have established a new link between microtubule-organizing centers and nuclear pores in budding yeast by unveiling an interaction between the Bfa1/Bub2 complex, a mitotic exit inhibitor that localizes on the spindle pole bodies, and the Nup159 nucleoporin. Bfa1/Bub2 association with Nup159 is reduced in metaphase to not interfere with proper spindle positioning. However, their interaction is stimulated in anaphase and assists the Nup159-dependent autophagy pathway. The asymmetric localization of Bfa1/Bub2 during mitosis raises the possibility that its interaction with Nup159 could differentially promote Nup159-mediated autophagic processes, which might be relevant for the maintenance of the replicative lifespan.

Pan-Cancer analysis and experimental validation identify the oncogenic nature of ESPL1: Potential therapeutic target in colorectal cancer.

Introduction: Extra spindle pole bodies like 1 (ESPL1) are required to continue the cell cycle, and its primary role is to initiate the final segregation of sister chromatids. Although prior research has revealed a link between ESPL1 and the development of cancer, no systematic pan-cancer analysis has been conducted. Combining multi-omics data with bioinformatics, we have thoroughly described the function of ESPL1 in cancer. In addition, we examined the impact of ESPL1 on the proliferation of numerous cancer cell lines. In addition, the connection between ESPL1 and medication sensitivity was verified using organoids obtained from colorectal cancer patients. All these results confirm the oncogene nature of ESPL1. Methods: Herein, we downloaded raw data from numerous publicly available databases and then applied R software and online tools to explore the association of ESPL1 expression with prognosis, survival, tumor microenvironment, tumor heterogeneity, and mutational profiles. To validate the oncogene nature of ESPL1, we have performed a knockdown of the target gene in various cancer cell lines to verify the effect of ESPL1 on proliferation and migration. In addition, patients' derived organoids were used to verify drug sensitivity. Results: The study found that ESPL1 expression was markedly upregulated in tumorous tissues compared to normal tissues, and high expression of ESPL1 was significantly associated with poor prognosis in a range of cancers. Furthermore, the study revealed that tumors with high ESPL1 expression tended to be more heterogeneous based on various tumor heterogeneity indicators. Enrichment analysis showed that ESPL1 is involved in mediating multiple cancer-related pathways. Notably, the study found that interference with ESPL1 expression significantly inhibited the proliferation of tumor cells. Additionally, the higher the expression of ESPL1 in organoids, the greater the sensitivity to PHA-793887, PAC-1, and AZD7762. Discussion: Taken together, our study provides evidence that ESPL1 may implicate tumorigenesis and disease progression across multiple cancer types, highlighting its potential utility as both a prognostic indicator and therapeutic target.

Ultrastructure expansion microscopy reveals the cellular architecture of budding and fission yeast.

The budding and fission yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have served as invaluable model organisms to study conserved fundamental cellular processes. Although super-resolution microscopy has in recent years paved the way to a better understanding of the spatial organization of molecules in cells, its wide use in yeasts has remained limited due to the specific know-how and instrumentation required, contrasted with the relative ease of endogenous tagging and live-cell fluorescence microscopy. To facilitate super-resolution microscopy in yeasts, we have extended the ultrastructure expansion microscopy (U-ExM) method to both S. cerevisiae and S. pombe, enabling a 4-fold isotropic expansion. We demonstrate that U-ExM allows imaging of the microtubule cytoskeleton and its associated spindle pole body, notably unveiling the Sfi1p-Cdc31p spatial organization on the appendage bridge structure. In S. pombe, we validate the method by monitoring the homeostatic regulation of nuclear pore complex number through the cell cycle. Combined with NHS-ester pan-labelling, which provides a global cellular context, U-ExM reveals the subcellular organization of these two yeast models and provides a powerful new method to augment the already extensive yeast toolbox. This article has an associated First Person interview with Kerstin Hinterndorfer and Felix Mikus, two of the joint first authors of the paper.

Phosphosites of the yeast centrosome component Spc110 contribute to cell cycle progression and mitotic exit.

Spc110 is an essential component of the spindle pole body (SPB), the yeast equivalent of the centrosome, that recruits the γ-tubulin complex to the nuclear side of the SPB to produce the microtubules that form the mitotic spindle. Here, we identified phosphosites S11 and S36 in maternally originated Spc110 and explored their functions in vivo. Yeast expressing non-phosphorylatable Spc110S11A had a distinct spindle phenotype characterised by higher levels of α-tubulin, which was frequently asymmetrically distributed between the two SPBs. Furthermore, expression of the double mutant Spc110S11AS36A had a delayed cell cycle progression. Specifically, the final steps of mitosis were delayed in Spc110S11AS36A cells, including expression and degradation of the mitotic cyclin Clb2, disassembling the mitotic spindle and re-localizing Cdc14 to the nucleoli, resulting in late mitotic exit and entry in G1. Thus, we propose that Spc110 phosphorylation at S11 and S36 is required to regulate timely cell cycle progression in budding yeast. This article has an associated First Person interview with the first author of the paper.

Human SFI1 and Centrin form a complex critical for centriole architecture and ciliogenesis.

Over the course of evolution, the centrosome function has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, with the presence of centrioles in humans and a spindle pole body (SPB) in yeast. Similarly, the composition of these two core elements has diverged, with the exception of Centrin and SFI1, which form a complex in yeast to initiate SPB duplication. However, it remains unclear whether this complex exists at centrioles and whether its function has been conserved. Here, using expansion microscopy, we demonstrate that human SFI1 is a centriolar protein that associates with a pool of Centrin at the distal end of the centriole. We also find that both proteins are recruited early during procentriole assembly and that depletion of SFI1 results in the loss of the distal pool of Centrin, without altering centriole duplication. Instead, we show that SFI1/Centrin complex is essential for centriolar architecture, CEP164 distribution, and CP110 removal during ciliogenesis. Together, our work reveals a conserved SFI1/Centrin module displaying divergent functions between mammals and yeast.

The cytoplasmic microtubule array in Neurospora crassa depends on microtubule-organizing centers at spindle pole bodies and microtubule +end-depending pseudo-MTOCs at septa.

γ-Tubulin ring complexes (γ-TuRC) mediate nucleation and anchorage of microtubules (MTs) to microtubule organizing centers (MTOCs). In fungi, the spindle pole body (SPB) is the functional equivalent of the centrosome, which is the main MTOC. In addition, non-centrosomal MTOCs (ncMTOCs) contribute to MT formation in some fungi like Schizosaccharomyces pombe and Aspergillus nidulans. In A. nidulans, MTOCs are anchored at septa (sMTOC) and share components of the outer plaque of the SPB. Here we show that the Neurospora crassa SPB is embedded in the nuclear envelope, with the γ-TuRC targeting proteins PCP-1Pcp1/PcpA located at the inner plaque and APS-2Mto1/ApsB located at the outer plaque of the SPB. PCP-1 was a specific component of nuclear MTOCs, while APS-2 was also present at the septal pore. Although γ-tubulin was only detected at the nucleus, spontaneous MT nucleation occurred in the apical and subapical cytoplasm during recovery from benomyl-induced MT depolymerization experiments. There was no evidence for MT nucleation at septa. However, without benomyl treatment MT plus-ends were organized in the septal pore through MTB-3EB1. Those septal MT plus ends polymerized MTs from septa in interphase cells Thus we conclude that the SPB is the only MT nucleation site in N. crassa, but the septal pore aids the MT network arrangement through the anchorage of the MT plus-ends through a pseudo-MTOC.

Spindle pole body component 25 and platelet-derived growth factor mediate crosstalk between tumor-associated macrophages and prostate cancer cells.

Tumor-associated macrophages (TAMs) are involved in the growth of prostate cancer (PrC), while the molecular mechanisms underlying the interactive crosstalk between TAM and PrC cells remain largely unknown. Platelet-derived growth factor (PDGF) is known to promote mesenchymal stromal cell chemotaxis to the tumor microenvironment. Recently, activation of spindle pole body component 25 (SPC25) has been shown to promote PrC cell proliferation and is associated with PrC stemness. Here, the relationship between SPC25 and PDGF in the crosstalk between TAM and PrC was investigated. Significant increases in both PDGF and SPC25 levels were detected in PrC specimens compared to paired adjacent normal prostate tissues. A significant correlation was detected between PDGF and SPC25 levels in PrC specimens and cell lines. SPC25 increased PDGF production and tumor cell growth in cultured PrC cells and in xenotransplantation. Mechanistically, SPC25 appeared to activate PDGF in PrC likely through Early Growth Response 1 (Egr1), while the secreted PDGF signaled to TAM through PDGFR on macrophages and polarized macrophages, which, in turn, induced the growth of PrC cells likely through their production and secretion of transforming growth factor ß1 (TGFß1). Thus, our data suggest that SPC25 triggers the crosstalk between TAM and PrC cells via SPC25/PDGF/PDGFR/TGFß1 receptor signaling to enhance PrC growth.

SIN-Like Pathway Kinases Regulate the End of Mitosis in the Methylotrophic Yeast Ogataea polymorpha.

The mitotic exit network (MEN) is a conserved signalling pathway essential for the termination of mitosis in the budding yeast Saccharomyces cerevisiae. All MEN components are highly conserved in the methylotrophic budding yeast Ogataea polymorpha, except for Cdc15 kinase. Instead, we identified two essential kinases OpHcd1 and OpHcd2 (homologue candidate of ScCdc15) that are homologous to SpSid1 and SpCdc7, respectively, components of the septation initiation network (SIN) of the fission yeast Schizosaccharomyces pombe. Conditional mutants for OpHCD1 and OpHCD2 exhibited significant delay in late anaphase and defective cell separation, suggesting that both genes have roles in mitotic exit and cytokinesis. Unlike Cdc15 in S. cerevisiae, the association of OpHcd1 and OpHcd2 with the yeast centrosomes (named spindle pole bodies, SPBs) is restricted to the SPB in the mother cell body. SPB localisation of OpHcd2 is regulated by the status of OpTem1 GTPase, while OpHcd1 requires the polo-like kinase OpCdc5 as well as active Tem1 to ensure the coordination of mitotic exit (ME) signalling and cell cycle progression. Our study suggests that the divergence of molecular mechanisms to control the ME-signalling pathway as well as the loss of Sid1/Hcd1 kinase in the MEN occurred relatively recently during the evolution of budding yeast.

Quantitative analysis of nuclear pore complex organization in Schizosaccharomyces pombe.

The number, distribution, and composition of nuclear pore complexes (NPCs) in the nuclear envelope varies between cell types and changes during cellular differentiation and in disease. To understand how NPC density and organization are controlled, we analyzed the NPC number and distribution in the fission yeast Schizosaccharomyces pombe using structured illumination microscopy. The small size of yeast nuclei, genetic features of fungi, and our robust image analysis pipeline allowed us to study NPCs in intact nuclei under multiple conditions. Our data revealed that NPC density is maintained across a wide range of nuclear sizes. Regions of reduced NPC density are observed over the nucleolus and surrounding the spindle pole body (SPB). Lem2-mediated tethering of the centromeres to the SPB is required to maintain NPC exclusion near SPBs. These findings provide a quantitative understanding of NPC number and distribution in S. pombe and show that interactions between the centromere and the nuclear envelope influences local NPC distribution.

Spindle pole body component 24 homolog potentiates tumor progression via regulation of SRY-box transcription factor 2 in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common pathological subtype of human kidney cancer with a high probability of metastasis. To understand the molecular processing essential for ccRCC tumorigenicity, we conducted an integrative in silico analysis of The Cancer Genome Atlas (TCGA) ccRCC dataset and clustered randomly interspersed short palindromic repeats (CRISPR) screening dataset of ccRCC cell lines from Depmap. We identified spindle pole body component 24 homolog (SPC24) as an essential gene for ccRCC cell lines with prognostic significance in the TCGA database. Targeting SPC24 by CRISPR/Cas9-mediated gene knockout attenuated ccRCC proliferation, metastasis, and in vivo tumor growth. Furthermore, we found that SPC24 regulates metastasis genes expression in a SRY-box transcription factor 2 (SOX2)-dependent manner. The anti-proliferative effects of SPC24 knockout were strengthened with SOX2 knockdown. Collectively, our findings suggest SPC24 has a pivotal function in promoting ccRCC progression, providing a new insight for the treatment of ccRCC.

Pcp1/pericentrin controls the SPB number in fission yeast meiosis and ploidy homeostasis.

During sexual reproduction, the zygote must inherit exactly one centrosome (spindle pole body [SPB] in yeasts) from the gametes, which then duplicates and assembles a bipolar spindle that supports the subsequent cell division. Here, we show that in the fission yeast Schizosaccharomyces pombe, the fusion of SPBs from the gametes is blocked in polyploid zygotes. As a result, the polyploid zygotes cannot proliferate mitotically and frequently form supernumerary SPBs during subsequent meiosis, which leads to multipolar nuclear divisions and the generation of extra spores. The blockage of SPB fusion is caused by persistent SPB localization of Pcp1, which, in normal diploid zygotic meiosis, exhibits a dynamic association with the SPB. Artificially induced constitutive localization of Pcp1 on the SPB is sufficient to cause blockage of SPB fusion and formation of extra spores in diploids. Thus, Pcp1-dependent SPB quantity control is crucial for sexual reproduction and ploidy homeostasis in fission yeast.

Localization of the ubiquitin ligase Dma1 to the fission yeast contractile ring is modulated by phosphorylation.

The timing of cytokinesis relative to other mitotic events in the fission yeast Schizosaccharomyces pombe is controlled by the septation initiation network (SIN). During a mitotic checkpoint, the SIN is inhibited by the E3 ubiquitin ligase Dma1 to prevent chromosome mis-segregation. Dma1 dynamically localizes to spindle pole bodies (SPBs) and the contractile ring (CR) during mitosis, though its role at the CR is unknown. Here, we examined whether Dma1 phosphorylation affects its localization or function. We found that preventing Dma1 phosphorylation by substituting the six phosphosites with alanines diminished its CR localization but did not affect its mitotic checkpoint function. These studies reinforce the conclusion that Dma1 localization to the SPB is key to its role in the mitotic checkpoint.

Loss of kinesin-8 improves the robustness of the self-assembled spindle in Schizosaccharomyces pombe.

Chromosome segregation in female meiosis in many metazoans is mediated by acentrosomal spindles, the existence of which implies that microtubule spindles self-assemble without the participation of the centrosomes. Although it is thought that acentrosomal meiosis is not conserved in fungi, we recently reported the formation of self-assembled microtubule arrays, which were able to segregate chromosomes, in fission yeast mutants, in which the contribution of the spindle pole body (SPB; the centrosome equivalent in yeast) was specifically blocked during meiosis. Here, we demonstrate that this unexpected microtubule formation represents a bona fide type of acentrosomal spindle. Moreover, a comparative analysis of these self-assembled spindles and the canonical SPB-dependent spindle reveals similarities and differences; for example, both spindles have a similar polarity, but the location of the γ-tubulin complex differs. We also show that the robustness of self-assembled spindles can be reinforced by eliminating kinesin-8 family members, whereas kinesin-8 mutants have an adverse impact on SPB-dependent spindles. Hence, we consider that reinforced self-assembled spindles in yeast will help to clarify the molecular mechanisms behind acentrosomal meiosis, a crucial step towards better understanding gametogenesis.

The role of Aspergillus nidulans polo-like kinase PlkA in microtubule-organizing center control.

Centrosomes are important microtubule-organizing centers (MTOC) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) have been described in many cell types. The functional analogs of centrosomes in fungi are the spindle pole bodies (SPBs). In Aspergillus nidulans, additional MTOCs have been discovered at septa (sMTOC). Although the core components are conserved in both MTOCs, their composition and organization are different and dynamic. Here, we show that the polo-like kinase PlkA binds the γ-tubulin ring complex (γ-TuRC) receptor protein ApsB and contributes to targeting ApsB to both MTOCs. PlkA coordinates the activities of the SPB outer plaque and the sMTOC. PlkA kinase activity was required for astral MT formation involving ApsB recruitment. PlkA also interacted with the γ-TuRC inner plaque receptor protein PcpA. Mitosis was delayed without PlkA, and the PlkA protein was required for proper mitotic spindle morphology, although this function was independent of its catalytic activity. Our results suggest that the polo-like kinase is a regulator of MTOC activities and acts as a scaffolding unit through interaction with γ-TuRC receptors.

Application of PALM Superresolution Microscopy to the Analysis of Microtubule-Organizing Centers (MTOCs) in Aspergillus nidulans.

Photoactivated localization microscopy (PALM), one of the super resolution microscopy methods improving the resolution limit to 20 nm, allows the detection of single molecules in complex protein structures in living cells. Microtubule-organizing centres (MTOCs) are large, multisubunit protein complexes, required for microtubule polymerization. The prominent MTOC in higher eukaryotes is the centrosome, and its functional ortholog in fungi is the spindle-pole body (SPB). There is ample evidence that besides centrosomes other MTOCs are important in eukaryotic cells. The filamentous ascomycetous fungus Aspergillus nidulans is a model organism, with hyphae consisting of multinucleate compartments separated by septa. In A. nidulans, besides the SPBs, a second type of MTOCs was discovered at septa (called septal MTOCs, sMTOC). All the MTOC components appear as big dots at SPBs and sMTOCs when tagged with a fluorescent protein and observed with conventional fluorescence microscopy due to the diffraction barrier. In this chapter, we describe the application of PALM in quantifying the numbers of individual proteins at both MTOC sites in A. nidulans and provide evidence that the composition of MTOCs is highly dynamic and dramatically changes during the cell cycle.