Regulation of Spermatogenesis: Differentiation of GFP-labeled Stem Cells and the Function of Cytoplasmic Bridges

During spermatogenesis, different genes are expressed in a strictly coordinated fashion providing an excellent model to study cell differentiation. Recent identification of testis specific genes and the development of green fluorescence protein (GFP) transgene technology and an in vivo system for studying the differentiation of transplanted male germ cells in infertile testis has opened new possibilities for studying the male germ cell differentiation at molecular level. We have employed these techniques in combination with transillumination based stage recognition (Parvinen and Vanha-Perttula, 1972) and squash preparation techniques (Parvinen and Hecht, 1981) to study the regulation of male germ cell differentiation. By using transgenic mice expressing enhanced-(E)GFP as a marker we have studied the expression and hormonal regulation of beta-actin and acrosin proteins in the developmentally different living male germ cells. Beta-actin was demonstrated in all male germ cells, whereas acrosin was expressed only in late meiotic and in postmeiotic cells. Follicle stimulating hormone stimulated b-actin-EGFP expression at stages I-VI and enhanced the formation of microtubules in spermatids and this way reduced the size of the acrosomic system. When EGFP expressing spermatogonial stem cells were transplanted into infertile mouse testis differentiation and the synchronized development of male germ cells could be observed during six months observation time. Each colony developed independently and maintained typical stage-dependent cell associations. Furthermore, if more than two colonies were fused, each of them was adjusted to one stage and synchronized. By studying living spermatids we were able to demonstrate novel functions for Golgi complex and chromatoid body in material sharing between neighbor spermatids. Immunosytochemical analyses revealed a transport of haploid cell specific proteins in spermatids (TRA54 and Shippo1) and through the intercellular bridges (TRA54). Cytoskeleton inhibitor (nocodazole) demonstrated the importance of microtubules in material sharing between spermatids and in preserving the integrity of the chromatoid body. Golgi complex inhibitor, brefeldin A, revealed the great importance of Golgi complex i) in acrosomic system formation ii) TRA54 translation and in iii) granule trafficking between spermatids.

The Chromatoid body entourage: Molecular characterization of the Chromatoid body‐associated cytoplasmic vesicles

Spermatogenesis is a unique process compared to cell differentiation in somatic tissues. Germ cells undergo a considerable number of metabolic and morphological changes during their differentiation: they initially proliferate by mitosis to increase in number; at some point they scramble their genetic material by meiosis, to create new genetic combinations that are the basis for evolution through natural selection and, finally, they change their shape and produce specialized structures characteristic of the mature sperm. Germ cells display an astonishingly broad transcription of their genome compared to differentiated somatic cells. Moreover, the different RNAs need to be specifically regulated in space and time for sperm production to occur appropriately. Different proteins localized in specific subcellular compartments, along with regulatory small RNAs, have an essential role in the proper execution of the different steps of spermatogenesis. These ribonucleoprotein granules interact with cytoplasmic vesicles and organelles to accomplish their role during sperm development. In this study, we characterized the most prominent ribonucleoprotein granule found in germ cells, the Chromatoid body (CB). For the first time we investigated the interaction of the CB with the cytoplasmic vesicles that surround it. These studies directed us to the description of Retromer proteins in germ cells and their involvement with the CB and the acrosome formation. Moreover, we discovered the interplay between the CB and the lysosome system in haploid round spermatids, and identified FYCO1, a new protein central to this interaction. Our results suggest that the vesicular transport system participates in the CB-mediated RNA regulation during sperm development.

β1 integrin regulation

Integrins are heterodimeric adhesion receptors mediating adhesion to extracellular matrix proteins and to other cells. Integrins are important in embryonic development, structural integrity of connective tissue, blood thrombus formation, and immune defense system. Integrins are transmembrane proteins whose ligand binding capacity (activity) is regulated by large conformational changes. Extracellular ligand binding or intracellular effector binding to integrin cytoplasmic face regulate integrin activity. Integrins are thus able to mediate bi-directional signaling. Integrin function is also regulated by intracellular location. Integrins are constantly recycled from endocytic vesicles to plasma membrane, and this has been shown to be important for cell migration and invasion as well. Deregulation of integrin functionality can lead to deleterious illnesses, such as bleeding or inflammatory disorders. It is also evident that integrin deregulation is associated with cancer progression. In this study, a novel Beta1 integrin associating protein, Rab21, was characterized. Rab21 binding to integrin cytoplasmic tail was shown to be important for Beta1 integrin endo- and exocytosis – intracellular trafficking. It was furher shown that this interaction has an important role in cell adhesion, migration, as well as in the final step of cell division, cytokinesis. This work showed that abrogation of Rab21 function or β1 integrin endocytic traffic, can lead to defects in cell division and results in formation of multinucleated cells. Multinucleation and especially tetraploidy can be a transient pathway to aneuploidy and tumorigenesis. This work characterized chromosomal deletions in rab21 locus in ovarian and prostate cancer samples and showed that a cell line with rab21 deletion also had impairment in cell division, which could be rescued by Rab21 re-expression. The work demonstrates an important role for Rab21 and Beta1 integrin traffic regulation in cell adhesion and division, and suggests a probable associaton with tumorigenesis. In this study, Beta1 integrin activity regulation was also addressed. A novel cell array platform for genome-scale RNAi screenings was characterized here. More than 4500 genes were knocked-down in prostate cancer cells using siRNA-mediated silencing. The effects on Beta1 integrin activity were analyzed upon knock-downs. The screen identified more that 400 putative regulators of Beta1 integrin activity in prostate cancer. In conclusion, this work will help us to understand complex regulatory pathways involved in cancer cell adhesion and migration.

The Integrin Tail: A Tale of Cell Motility and Division

Integrin transmembrane receptor functions are regulated by adaptor molecules binding to their alpha and beta subunit intracellular domains, or tails, thus affecting integrin traffic and adhesion during e.g. cell motility. Interestingly, many cellular proteins function in both cell motility and cell division, thus raising the possibility that integrins might be involved in regulating the cell cycle. A thorough understanding of cell division is essential in cell biology and in human malignancies. It is well established that failures to complete cell cycle can give rise to genetically unstable cells with tumorigenic properties. Transformed cells promote the disruption of intercellular adhesions such as tight junctions, and this correlates with the onset of cell motility, invasion and unfavorable prognosis in cancer. In this study, we analyzed integrin regulation, mediated by adaptor binding to the  subunit tail, during cell motility and cell division. We revealed a novel molecular mechanism by which Rab21, through association with the integrin alpha subunits, drives integrin endosomal traffic during mitotic phases. In addition, we found indications for this finding in vivo, as RAB21 gene deletions were mapped in ovarian and prostate cancer samples. Importantly, the multinucleated phenotype of cultured ovarian cancer cells could be reverted by Rab21 overexpression. In this thesis work, we also show how the tight junction protein ZO-1 unexpectedly interacts with the 5 integrin cytoplasmic domain in the lamellipodia to promote cell motility and at the cleavage furrow to support separation of the daughter cells. The alpha5-ZO-1 complex formation was dependent on PKC which regulates ZO-1 phosphorylation and its subcellular localization. In addition, by an in situ detection method, we showed that a subset of metastatic human lung cancers expressed the alpha5beta-ZO-1 complex. Taken together, we were able to identify new molecular pathways that regulate integrin functions in an alpha tail-mediated fashion. These findings firmly suggest that genetic alterations in integrin traffic may lead to progression of tumorigenesis as a result of failed cell division. Also, the interplay of integrins and ZO-1 in forming spatially regulated adhesive structures broadens our view of crosstalk between pathways and distinct adhesive structures that can be involved in cancer cell biology.

Novel Players in the Integrin Signaling Orchestra: TCPTP and MDGI

Metastases are the major cause of cancer deaths. Tumor cell dissemination from the primary tumor utilizes dysregulated cellular adhesion and upregulated proteolytic degradation of the extracellular matrix for progeny formation in distant organs. Integrins are transmembrane adhesive receptors mediating cellcell and cellmatrix interactions that are crucial for regulating cell migration, invasion, proliferation, and survival. Consequently, increased integrin activity is associated with augmented migration and invasion capacity in several cancer types. Heterodimeric integrins consist of an alpha - and beta-subunit that are held together in a bent conformation when the receptor is inactive, but extension and separation of subdomains is observed during receptor activation. Either inside-out or outside-in activation of receptors is possible through the intracellular molecule binding to an integrin cytoplasmic domain or extracellular ligand association with an integrin ectodomain, respectively. Several regulatory binding partners have been characterized for integrin cytoplasmic beta-domains, but the regulators interacting with the cytoplasmic alpha-domains have remained elusive. In this study, we performed yeast two-hybrid screens to identify novel binding partners for the cytoplasmic integrin alpha-domains. Further examination of two plausible candidates revealed a significant coregulatory role of an integrin alpha-subunit for cellular signaling processes. T-cell protein tyrosine phosphatase (TCPTP) showed a specific interaction with the cytoplasmic tail of integrin alpha1. This association stimulated TCPTP phosphatase activity, leading to negative regulation of epidermal growth factor receptor (EGFR) signaling and diminished anchorage-independent growth. Another candidate, mammary-derived growth inhibitor (MDGI), exhibited binding to several different integrin cytoplasmic alpha-tails through a conserved GFFKR sequence. MDGI overexpression in breast cancer cells altered EGFR trafficking and caused a remarkable accumulation of EGFR in the cytoplasm. We further demonstrated in vivo that MDGI expression induced a novel form of anti-EGFR therapy resistance. Moreover, MDGI binding to α-tails retained integrin in an inactive conformation attenuating integrin-mediated adhesion, migration, and invasion. In agreement with these results, sustained MDGI expression in breast cancer patients correlated with an increased 10-year distant disease-free survival. Taken together, the integrin signaling network is far from a complete view and future work will doubtless broaden our understanding further.

Nuclear Matrix in Apoptotic Cell Death and Cell Proliferation. The role of Nuclear Mitotic Apparatus (NuMA) Protein

The nucleus is a membrane enclosed organelle containing most of the genetic information of the cell in the form of chromatin. The nucleus, which can be divided into many sub-organelles such as the nucleoli, the Cajal bodies and the nuclear lamina, is the site for several essential cellular functions such as the DNA replication and its regulation and most of the RNA synthesis and processing. The nucleus is often affected in disease: the size and the shape of the nucleus, the chromatin distribution and the size of the nucleoli have remained the basis for the grading of several cancers. The maintenance of the vertebrate body shape depends on the skeleton. Similarly, in a smaller context, the shape of the cell and the nucleus are mainly regulated by the cytoskeletal and nucleoskeletal elements. The nuclear matrix, which by definition is a detergent, DNase and salt resistant proteinaceous nuclear structure, has been suggested to form the nucleoskeleton responsible for the nuclear integrity. Nuclear mitotic apparatus protein, NuMA, a component of the nuclear matrix, is better known for its mitotic spindle organizing function. NuMA is one of the nuclear matrix proteins suggested to participate in the maintenance of the nuclear integrity during interphase but its interphase function has not been solved to date. This thesis study concentrated on the role of NuMA and the nuclear matrix as structural and functional components of the interphase nucleus. The first two studies clarified the essential role of caspase-3 in the disintegration of the nuclear structures during apoptosis. The second study also showed NuMA and chromatin to co-elute from cells in significant amounts and the apoptotic cleavage of NuMA was clarified to have an important role in the dissociation of NuMA from the chromatin. The third study concentrated on the interphase function of NuMA showing NuMA depletion to result in cell cycle arrest and the cytoplasmic relocalization of NuMA interaction partner GAS41. We suggest that the relocalization of the transcription factor GAS41 may mediate the cell cycle arrest. Thus, this study has given new aspects in the interactions of NuMA, chromatin and the nuclear matrix.

Fluorescence-based imaging of cellular defect in lysinuric protein intolerance (LPI)

Fluoresenssiperusteiset kuvantamismenetelmät lysinurisen proteiini-intoleranssin (LPI) soluhäiriön tutkimuksessa Lysinurinen proteiini-intoleranssi on suomalaiseen tautiperintöön kuuluva autosomaalisesti peit¬tyvästi periytyvä sairaus, jonka aiheuttaa kationisten aminohappojen kuljetushäiriö munuaisten ja ohutsuolen epiteelisolujen basolateraalikalvolla. Aminohappojen kuljetushäiriö johtaa moniin oirei¬siin, kuten kasvuhäiriöön, osteoporoosiin, immuunijärjestelmän häiriöihin, oksenteluun ja runsaspro¬teiinisen ravinnon nauttimisen jälkeiseen hyperammonemiaan. LPI-geeni SLC7A7 (solute carrier family 7 member 7) koodaa y+LAT1 proteiinia, joka on basolateraali¬nen kationisten ja neutraalien aminohappojen kuljettimen kevyt ketju, joka muodostaa heterodimee¬rin raskaan alayksikön 4F2hc:n kanssa. Tällä hetkellä SLC7A7-geenistä tunnetaan yli 50 LPI:n aiheut¬tavaa mutaatiota. Tässä tutkimuksessa erityyppisiä y+LAT1:n LPI-mutaatiota sekä yhdeksän C-terminaalista polypep¬tidiä lyhentävää deleetiota kuvannettiin nisäkässoluissa y+LAT1:n GFP (green fluorescent protein) -fuusioproteiineina. Tulokset vahvistivat muissa soluissa tehdyt havainnot siitä, että 4F2hc on edel¬lytyksenä y+LAT1:n solukalvokuljetukselle, G54V-pistemutantti sijaitsee solukalvolla samoin kuin vil¬lityyppinen proteiini, mutta lukukehystä muuttavia ja proteiinia lyhentäviä mutantteja ei kuljeteta solukalvoon. Lisäksi havaittiin, että poikkeuksena tästä säännöstä ovat y+LAT1-deleetioproteiinit, joista puuttui korkeintaan 50 C-terminaalista aminohappoa. Nämä lyhentyneet kuljettimet sijaitsevat solukalvolla kuten villityyppiset ja LPI-pistemutanttiproteiinit. Dimerisaation osuutta kuljetushäiriön synnyssä tutkittiin käyttämällä fluorescence resonance energy transfer (FRET) menetelmää. Heterodimeerin alayksiköistä kloonattiin ECFP (cyan) ja EYFP (yellow) fuusioproteiinit, joita ilmennettiin nisäkässoluissa, ja FRET mitattiin virtaussytometri-FRET -menetel¬mällä (FACS-FRET). Tutkimuksissa kaikkien mutanttien havaittiin dimerisoituvan yhtä tehokkaasti. Kul¬jetushäiriön syynä ei siten ole alayksiköiden dimerisaation estyminen mutaation seurauksena. Tutkimuksessa havaittiin, että kaikki mutantti-y+LAT1-transfektiot tuottavat vähemmän transfektoi¬tuneita soluja kuin villityyppisen y+LAT1:n transfektiot. Solupopulaatioissa, joihin oli tranfektoitu lu¬kukehystä muuttava tai stop-kodonin tuottava mutaatio havaittiin suurempi kuolleisuus kuin saman näytteen transfektoitumattomissa soluissa, kun taas villityyppistä tai G54V-pistemutanttia tuottavas¬sa solupopulaatiossa oli pienempi kuolleisuus kuin saman näytteen fuusioproteiinia ilmentämättö¬missä soluissa. Tulos osoittaa mutanttiproteiinien erilaiset vaikutukset niitä ilmentäviin soluihin, joko suoraan y+LAT1:n tai 4F2hc:n kautta aiheutuneina. LPIFin SLC7A7 lähetti-RNA:n määrä ei merkittävästi poikennut villityyppisen määrästä fibroblasteissa ja lymfoblasteissa. SLC7A7:n promoottorianalyysissä oli osoitettavissa säätelyalueita geenin 5’ ei-koo¬daavalla alueella sekä ensimmäisten kahden intronin alueella. LPI-taudin tautimekanismin kannalta keskeisin tekijä on kuitenkin aminohappokuljetuksen häiriö, jonka vaikutuksesta näistä aminohapoista riippuvaiset prosessit elimistössä eivät toimi normaalisti. Havaittu virheellinen y+LAT1/4F2hc kuljetuskompleksin sijainti edellyttää lisätutkimuksia sen mahdol¬lisen kliinisen merkityksen selvittämiseksi.

Chromatoid body mediated RNA regulation in mouse male germline

Male germ cell differentiation, spermatogenesis is an exceptional developmental process that produces a massive amount of genetically unique spermatozoa. The complexity of this process along with the technical limitations in the germline research has left many aspects of spermatogenesis poorly understood. Post-meiotic haploid round spermatids possess the most complex transcriptomes of the whole body. Correspondingly, efficient and accurate control mechanisms are necessary to deal with the huge diversity of transcribed RNAs in these cells. The high transcriptional activity in round spermatids is accompanied by the presence of an uncommonly large cytoplasmic ribonucleoprotein granule, called the chromatoid body (CB) that is conjectured to participate in the RNA post-transcriptional regulation. However, very little is known about the possible mechanisms of the CB function. The development of a procedure to isolate CBs from mouse testes was this study’s objective. Anti-MVH immunoprecipitation of cross-linked CBs from a fractionated testicular cell lysate was optimized to yield considerable quantities of pure and intact CBs from mice testes. This protocol produced reliable and reproducible data from the subsequent analysis of CB’s protein and RNA components. We found that the majority of the CB’s proteome consists of RNA-binding proteins that associate functionally with different pathways. We also demonstrated notable localization patterns of one of the CB transient components, SAM68 and showed that its ablation does not change the general composition or structure of the CB. CB-associated RNA analysis revealed a strong accumulation of PIWI-interacting RNAs (piRNAs), mRNAs and long non-coding RNAs (lncRNAs) in the CB. When the CB transcriptome and proteome analysis results were combined, the most pronounced molecular functions in the CB were related to piRNA pathway, RNA post-transcriptional processing and CB structural scaffolding. In addition, we demonstrated that the CB is a target for the main RNA flux from the nucleus throughout all steps of round spermatid development. Moreover, we provided preliminary evidence that those isolated CBs slice target RNAs in vitro in an ATPdependent manner. Altogether, these results make a strong suggestion that the CB functions involve RNA-related and RNA-mediated mechanisms. All the existing data supports the hypothesis that the CB coordinates the highly complex haploid transcriptome during the preparation of the male gametes for fertilization. Thereby, this study provides a fundamental basis for the future functional analyses of ribonucleoprotein granules and offers also important insights into the mechanisms governing male fertility.

Roles of novel biomarkers in progression of cutaneous squamous cell carcinoma

Roles of novel biomarkers was studied in progression of cutaneous squamous cell carcinoma (cSCC) as the most common metastatic skin cancer. The incidence of cSCC is increasing worldwide due to lifestyle changes such as recreational exposure to sunlight and the aging of the population. Because of an emerging need for molecular markers for the progression of cSCC, we set our goal to characterize three distinct novel markers overexpressed in cSCC cells. Our results identified overexpression of serpin peptidase inhibitor clade A member 1 (SerpinA1), EphB2 and absent in melanoma 2 (AIM2) in cSCC cell lines compared with normal human epidermal keratinocytes (NHEKs). Immunohistochemical analysis of SerpinA1, EphB2 and AIM2 revealed abundant tumor cell-specific expression of cytoplasmic SerpinA1 and AIM2 and cytoplasmic and membranous EphB2 in cSCC tumors in vivo. The staining intensity of SerpinA1, EphB2 and AIM2 was significantly stronger in cSCC as compared with carcinoma in situ (cSCCIS) and actinic keratosis (AK). Tumor cell-associated SerpinA1 and EphB2 was noted in chemically induced mouse skin SCC, and the staining intensity was stronger in mouse cSCCs than in untreated skin. AIM2 staining intensity was significantly more abundant in cSCC of organ transplant recipients (OTR) than in sporadic cSCC in vivo. EphB2 knockdown resulted in inhibition of migration in cSCC cells. In addition, knockdown of EphB2 and AIM2 was found to inhibit the proliferation and invasion of cSCC cells and to delay the growth and vascularization of cSCC xenografts in vivo. Altogether, these findings identify SerpinA1 as a novel biomarker for cSCC. In addition, characterization of the roles of EphB2 and AIM2 in the progression of cSCC was implicated them as possible therapeutic targets for the treatment of cSCC particularly in unresectable and metastatic tumors.

Models to study intermediate filament dynamics and functions

Intermediate filaments are part of the cytoskeleton and nucleoskeleton; they provide cells with structure and have important roles in cell signalling. The IFs are a large protein family with more than 70 members; each tightly regulated and expressed in a cell type-specific manner. Although the IFs have been known and studied for decades, our knowledge about their specific functions is still limited, despite the fact that mutations in IF genes cause numerous severe human diseases. In this work, three IF proteins are examined more closely; the nuclear lamin A/C and the cytoplasmic nestin and vimentin. In particular the regulation of lamin A/C dynamics, the role of nestin in muscle and body homeostasis as well as the functions and evolutionary aspects of vimentin are investigated. Together this data highlights some less well understood functions of these IFs. We used mass-spectrometry to identify inter-phase specific phosphorylation sites on lamin A. With the use of genetically engineered lamin A protein in combination with high resolution microscopy and biochemical methods we discovered novel roles for this phosphorylation in regulation of lamin dynamics. More specifically, our data suggests that the phosphorylation of certain amino acids in lamin A determines the localization and dynamics of the protein. In addition, we present results demonstrating that lamin A regulates Cdk5-activity. In the second study we use mice lacking nestin to gain more knowledge of this seldom studied protein. Our results show that nestin is essential for muscle regeneration; mice lacking nestin recover more slowly from muscle injury and show signs of spontaneous muscle regeneration, indicating that their muscles are more sensitive to stresses and injury. The absence of nestin also leads to decreased over-all muscle mass and slower body growth. Furthermore, nestin has a role in controlling testicle homeostasis as nestin-/- male mice show a greater variation in testicle size. The common fruit fly Drosophila melanogaster lacks cytoplasmic IFs as most insects do. By creating a fly that expresses human vimentin we establish a new research platform for vimentin studies, as well as provide a new tool for the studies of IF evolution.