CD38: an ecto-enzyme with functional diversity in T cells.

CD38, a nicotinamide adenine dinucleotide (NAD)+ glycohydrolase, is considered an activation marker of T lymphocytes in humans that is highly expressed during certain chronic viral infections. T cells constitute a heterogeneous population; however, the expression and function of CD38 has been poorly defined in distinct T cell compartments. We investigated the expression and function of CD38 in naïve and effector T cell subsets in the peripheral blood mononuclear cells (PBMCs) from healthy donors and people with HIV (PWH) using flow cytometry. Further, we examined the impact of CD38 expression on intracellular NAD+ levels, mitochondrial function, and intracellular cytokine production in response to virus-specific peptide stimulation (HIV Group specific antigen; Gag). Naïve T cells from healthy donors showed remarkably higher levels of CD38 expression than those of effector cells with concomitant reduced intracellular NAD+ levels, decreased mitochondrial membrane potential and lower metabolic activity. Blockade of CD38 by a small molecule inhibitor, 78c, increased metabolic function, mitochondrial mass and mitochondrial membrane potential in the naïve T lymphocytes. PWH exhibited similar frequencies of CD38+ cells in the T cell subsets. However, CD38 expression increased on Gag-specific IFN-γ and TNF-α producing cell compartments among effector T cells. 78c treatment resulted in reduced cytokine production, indicating its distinct expression and functional profile in different T cell subsets. In summary, in naïve cells high CD38 expression reflects lower metabolic activity, while in effector cells it preferentially contributes to immunopathogenesis by increasing inflammatory cytokine production. Thus, CD38 may be considered as a therapeutic target in chronic viral infections to reduce ongoing immune activation.

Fluorescence Lifetime Imaging of Human Sub-RPE Calcification In Vitro Following Chlortetracycline Infusion.

We have shown that all sub-retinal pigment epithelial (sub-RPE) deposits examined contain calcium phosphate minerals: hydroxyapatite (HAP), whitlockite (Wht), or both. These typically take the form of ca. 1 µm diameter spherules or >10 µm nodules and appear to be involved in the development and progression of age-related macular degeneration (AMD). Thus, these minerals may serve as useful biomarkers the for early detection and monitoring of sub-RPE changes in AMD. We demonstrated that HAP deposits could be imaged in vitro by fluorescence lifetime imaging microscopy (FLIM) in flat-mounted retinas using legacy tetracycline antibiotics as selective sensors for HAP. As the contrast on a FLIM image is based on the difference in fluorescence lifetime and not intensity of the tetracycline-stained HAP, distinguishing tissue autofluorescence from the background is significantly improved. The focus of the present pilot study was to assess whether vascular perfusion of the well tolerated and characterized chlortetracycline (widely used as an orally bioavailable antibiotic) can fluorescently label retinal HAP using human cadavers. We found that the tetracycline delivered through the peripheral circulation can indeed selectively label sub-RPE deposits opening the possibility for its use for ophthalmic monitoring of a range of diseases in which deposit formation is reported, such as AMD and Alzheimer disease (AD).

Buffered EGFR signaling regulated by spitz-to-argos expression ratio is a critical factor for patterning the Drosophila eye.

The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays a critical role in regulating tissue patterning. Drosophila EGFR signaling achieves specificity through multiple ligands and feedback loops to finetune signaling outcomes spatiotemporally. The principal Drosophila EGF ligand, cleaved Spitz, and the negative feedback regulator, Argos are diffusible and can act both in a cell autonomous and non-autonomous manner. The expression dose of Spitz and Argos early in photoreceptor cell fate determination has been shown to be critical in patterning the Drosophila eye, but the exact identity of the cells expressing these genes in the larval eye disc has been elusive. Using single molecule RNA Fluorescence in situ Hybridization (smFISH), we reveal an intriguing differential expression of spitz and argos mRNA in the Drosophila third instar eye imaginal disc indicative of directional non-autonomous EGFR signaling. By genetically tuning EGFR signaling, we show that rather than absolute levels of expression, the ratio of expression of spitz-to-argos to be a critical determinant of the final adult eye phenotype. Proximate effects on EGFR signaling in terms of cell cycle and differentiation markers are affected differently in the different perturbations. Proper ommatidial patterning is robust to thresholds around a tightly maintained wildtype spitz-to-argos ratio, and breaks down beyond. This provides a powerful instance of developmental buffering against gene expression fluctuations.

An actomyosin clamp assembled by the Amphiphysin-Rho1-Dia/DAAM-Rok pathway reinforces somatic cell membrane folded around spermatid heads.

Membrane curvature recruits Bin-Amphiphysin-Rvs (BAR)-domain proteins and induces local F-actin assembly, which further modifies the membrane curvature and dynamics. The downstream molecular pathway in vivo is still unclear. Here, we show that a tubular endomembrane scaffold supported by contractile actomyosin stabilizes the somatic cyst cell membrane folded around rigid spermatid heads during the final stages of sperm maturation in Drosophila testis. The structure resembles an actin "basket" covering the bundle of spermatid heads. Genetic analyses suggest that the actomyosin organization is nucleated exclusively by the formins - Diaphanous and Dishevelled Associated Activator of Morphogenesis (DAAM) - downstream of Rho1, which is recruited by the BAR-domain protein Amphiphysin. Actomyosin activity at the actin basket gathers the spermatid heads into a compact bundle and resists the somatic cell invasion by intruding spermatids. These observations reveal a distinct response mechanism of actin-membrane interactions, which generates a cell-adhesion-like strategy through active clamping.

The HIV-1 Antisense Protein ASP Is a Transmembrane Protein of the Cell Surface and an Integral Protein of the Viral Envelope.

The negative strand of HIV-1 encodes a highly hydrophobic antisense protein (ASP) with no known homologs. The presence of humoral and cellular immune responses to ASP in HIV-1 patients indicates that ASP is expressed in vivo, but its role in HIV-1 replication remains unknown. We investigated ASP expression in multiple chronically infected myeloid and lymphoid cell lines using an anti-ASP monoclonal antibody (324.6) in combination with flow cytometry and microscopy approaches. At baseline and in the absence of stimuli, ASP shows polarized subnuclear distribution, preferentially in areas with low content of suppressive epigenetic marks. However, following treatment with phorbol 12-myristate 13-acetate (PMA), ASP translocates to the cytoplasm and is detectable on the cell surface, even in the absence of membrane permeabilization, indicating that 324.6 recognizes an ASP epitope that is exposed extracellularly. Further, surface staining with 324.6 and anti-gp120 antibodies showed that ASP and gp120 colocalize, suggesting that ASP might become incorporated in the membranes of budding virions. Indeed, fluorescence correlation spectroscopy studies showed binding of 324.6 to cell-free HIV-1 particles. Moreover, 324.6 was able to capture and retain HIV-1 virions with efficiency similar to that of the anti-gp120 antibody VRC01. Our studies indicate that ASP is an integral protein of the plasma membranes of chronically infected cells stimulated with PMA, and upon viral budding, ASP becomes a structural protein of the HIV-1 envelope. These results may provide leads to investigate the possible role of ASP in the virus replication cycle and suggest that ASP may represent a new therapeutic or vaccine target.IMPORTANCE The HIV-1 genome contains a gene expressed in the opposite, or antisense, direction to all other genes. The protein product of this antisense gene, called ASP, is poorly characterized, and its role in viral replication remains unknown. We provide evidence that the antisense protein, ASP, of HIV-1 is found within the cell nucleus in unstimulated cells. In addition, we show that after PMA treatment, ASP exits the nucleus and localizes on the cell membrane. Moreover, we demonstrate that ASP is present on the surfaces of viral particles. Altogether, our studies identify ASP as a new structural component of HIV-1 and show that ASP is an accessory protein that promotes viral replication. The presence of ASP on the surfaces of both infected cells and viral particles might be exploited therapeutically.

Atypical septate junctions maintain the somatic enclosure around maturing spermatids and prevent premature sperm release in Drosophila testis.

Tight junctions prevent paracellular flow and maintain cell polarity in an epithelium. These junctions are also required for maintaining the blood-testis barrier, which is essential for sperm differentiation. Septate junctions in insects are orthologous to the tight junctions. In Drosophila testis, major septate junction components co-localize at the interface of germline and somatic cells initially, and then condense between the two somatic cells in a cyst after germline meiosis. Their localization is extensively remodeled in subsequent stages. We find that characteristic septate junctions are formed between the somatic cyst cells at the elongated spermatid stage. Consistent with previous reports, knockdown of essential junctional components - Discs-large-1 and Neurexin-IV - during the early stages disrupted sperm differentiation beyond the spermatocyte stage. Knockdown of these proteins during the final stages of spermatid maturation caused premature release of spermatids inside the testes, resulting in partial loss of male fertility. These results indicate the importance of maintaining the integrity of the somatic enclosure during spermatid coiling and release in Drosophila testis. It also highlights the functional similarity with the tight junction proteins during mammalian spermatogenesis.This article has an associated First Person interview with the first author of the paper.

Somatic ERK activation during transit amplification is essential for maintaining the synchrony of germline divisions in Drosophila testis.

Transit amplification (TA) of progenitor cells maintains tissue homeostasis by balancing proliferation and differentiation. In Drosophila testis, the germline proliferation is tightly regulated by factors present in both the germline and the neighbouring somatic cyst cells (SCCs). Although the exact mechanism is unclear, the epidermal growth factor receptor (EGFR) activation in SCCs has been reported to control spermatogonial divisions within a cyst, through downstream activations of Rac1-dependent pathways. Here, we report that somatic activation of the mitogen-activated protein kinase (Rolled/ERK) downstream of EGFR is required to synchronize the mitotic divisions and regulate the transition to meiosis. The process operates independently of the Bag-of-marble activity in the germline. Also, the integrity of the somatic cyst enclosure is inessential for this purpose. Together, these results suggest that synchronization of germ-cell divisions through somatic activation of distinct ERK-downstream targets independently regulates TA and subsequent differentiation of neighbouring germline cells.

Targeting the Late Stage of HIV-1 Entry for Antibody-Dependent Cellular Cytotoxicity: Structural Basis for Env Epitopes in the C11 Region.

Antibodies can have an impact on HIV-1 infection in multiple ways, including antibody-dependent cellular cytotoxicity (ADCC), a correlate of protection observed in the RV144 vaccine trial. One of the most potent ADCC-inducing epitopes on HIV-1 Env is recognized by the C11 antibody. Here, we present the crystal structure, at 2.9 Å resolution, of the C11-like antibody N12-i3, in a quaternary complex with the HIV-1 gp120, a CD4-mimicking peptide M48U1, and an A32-like antibody, N5-i5. Antibody N12-i3 recognizes an epitope centered on the N-terminal "eighth strand" of a critical ß sandwich, which our analysis indicates to be emblematic of a late-entry state, after the gp120 detachment. In prior entry states, this sandwich comprises only seven strands, with the eighth strand instead pairing with a portion of the gp120 C terminus. The conformational gymnastics of HIV-1 gp120 thus includes altered ß-strand pairing, possibly to reduce immunogenicity, although nevertheless still recognized by the human immune system.

Molecular basis for epitope recognition by non-neutralizing anti-gp41 antibody F240.

Antibody-dependent cell-mediated cytotoxicity (ADCC) by non-neutralizing antibodies (nnAbs) specific to the HIV envelope (Env) glycoproteins present at the surface of virus sensitized or infected cells plays a role in the effective adaptive immune response to HIV. Here, we explore the molecular basis for the epitope at the disulfide loop region (DLR) of the principal immunodominant domain of gp41, recognized by the well-known nnAb F240. Our structural studies reveal details of the F240-gp41 interface and describe a structure of DLR that is distinct from known conformations of this region studied in the context of either CD4-unliganded Env trimer or the gp41 peptide in the unbound state. These data coupled with binding and functional analyses indicate that F240 recognizes non-trimeric Env forms which are significantly overexpressed on intact virions but poorly represented at surfaces of cells infected with infectious molecular clones and endogenously-infected CD4 T cells from HIV-1-infected individuals. Furthermore, although we detect ADCC activities of F240 against cells spinoculated with intact virions, our data suggest that these activities result from F240 recognition of gp41 stumps or misfolded Env variants present on virions rather than its ability to recognize functional gp41 transition structures emerging on trimeric Env post CD4 receptor engagement.

Localized, Reactive F-Actin Dynamics Prevents Abnormal Somatic Cell Penetration by Mature Spermatids.

Spermatogenesis occurs inside a somatic cell enclosure. Sperm release, the most important final step and a target for contraceptives, has been extensively studied in fixed tissue preparations. Here, we provide a time-lapse description of the release process in Drosophila testis ex vivo. We show that the spermatid tails exit the somatic enclosure and enter the testicular duct first, followed by the spermatid heads. Prior to this, individual spermatid heads attempt to invade the head cyst cell, and on each occasion they are repelled by a rapid and local F-actin polymerization response from the head cyst cell. The F-actin assembly involves N-WASp, D-WIP, and Arp2/3 complex and dissipates once the spermatid head retreats back into the fold. These findings revise the existing spermiation model in Drosophila and suggest that somatic cells can actively oppose mechanical cell invasion attempts using calibrated F-actin dynamics in situ.

A conditional Orco requirement in the somatic cyst cells for maintaining spermatids in a tight bundle in Drosophila testis.

Odorant receptors (OR) heterodimerizes with the OR co-receptor (Orco), forming specific odorant-gated cation channels, which are key to odor reception at the olfactory sensory neurons (OSN). Mammalian ORs are expressed in many other tissues, including testis. However, their biological implications are yet to be fully ascertained. In the mosquito, Orco is localized along the sperm tail and is indicated to maintain fidelity. Here, we show that orco expresses in Drosophila testis. The levels are higher in the somatic cyst cells. The orco-null mutants are perfectly fertile at 25 degree C. At 28 degree C, the coiled spermatid bundles are severely disrupted. The loss of Orco also disrupts the actin cap, which forms inside the head cyst cell at the rostral ends of the spermatid nuclei after coiling, and plays a key role in preventing the abnormal release of spermatids from the cyst enclosure. Both the defects are rescued by the somatic cyst cell-specific expression of the UAS-orco transgene. These results highlight a novel role of Orco in the somatic tissue during sperm release.

The C-terminal domain of the bacteriophage T4 terminase docks on the prohead portal clip region during DNA packaging.

Bacteriophage ATP-based packaging motors translocate DNA into a pre-formed prohead through a dodecameric portal ring channel to high density. We investigated portal-terminase docking interactions at specifically localized residues within a terminase-interaction region (aa279-316) in the phage T4 portal protein gp20 equated to the clip domain of the SPP1 portal crystal structure by 3D modeling. Within this region, three residues allowed A to C mutations whereas three others did not, consistent with informatics analyses showing the tolerated residues are not strongly conserved evolutionarily. About 7.5nm was calculated by FCS-FRET studies employing maleimide Alexa488 dye labeled A316C proheads and gp17 CT-ReAsH supporting previous work docking the C-terminal end of the T4 terminase (gp17) closer to the N-terminal GFP-labeled portal (gp20) than the N-terminal end of the terminase. Such a terminase-portal orientation fits better to a proposed "DNA crunching" compression packaging motor and to portal determined DNA headful cutting.

Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation.

Viral genome packaging into capsids is powered by high-force-generating motor proteins. In the presence of all packaging components, ATP-powered translocation in vitro expels all detectable tightly bound YOYO-1 dye from packaged short dsDNA substrates and removes all aminoacridine dye from packaged genomic DNA in vivo. In contrast, in the absence of packaging, the purified T4 packaging ATPase alone can only remove up to ∼1/3 of DNA-bound intercalating YOYO-1 dye molecules in the presence of ATP or ATP-γ-S. In sufficient concentration, intercalating dyes arrest packaging, but rare terminase mutations confer resistance. These distant mutations are highly interdependent in acquiring function and resistance and likely mark motor contact points with the translocating DNA. In stalled Y-DNAs, FRET has shown a decrease in distance from the phage T4 terminase C terminus to portal consistent with a linear motor, and in the Y-stem DNA compression between closely positioned dye pairs. Taken together with prior FRET studies of conformational changes in stalled Y-DNAs, removal of intercalating compounds by the packaging motor demonstrates conformational change in DNA during normal translocation at low packaging resistance and supports a proposed linear "DNA crunching" or torsional compression motor mechanism involving a transient grip-and-release structural change in B form DNA.

Dynein light chain 1 (LC8) association enhances microtubule stability and promotes microtubule bundling.

BACKGROUND: Dynein Light Chain 1 (LC8) has been shown to pull down tubulin subunits, suggesting that it interacts with microtubules. RESULTS: LC8 decorates microtubules in vitro and in Drosophila embryos, promotes microtubule assembly, and stabilizes microtubules both in vitro and in tissue-cultured cells. CONCLUSION: LC8 stabilizes microtubules. SIGNIFICANCE: Data provide the first evidence of a novel MAP-like function of LC8. Dynein light chain 1 (LC8), a highly conserved protein, is known to bind to a variety of different polypeptides. It functions as a dimer, which is inactivated through phosphorylation at the Ser-88 residue. A loss of LC8 function causes apoptosis in Drosophila embryos, and its overexpression induces malignant transformation of breast cancer cells. Here we show that LC8 binds to tubulin, promotes microtubule assembly, and induces the bundling of reconstituted microtubules in vitro. Furthermore, LC8 decorates microtubules both in Drosophila embryos and in HeLa cells, increases the microtubule stability, and promotes microtubule bundling in these cells. Microtubule stability influences a number of different cellular functions including mitosis and cell differentiation. The LC8 overexpression reduces the susceptibility of microtubules to cold and nocodazole-induced depolymerization in tissue-cultured cells and increases microtubule acetylation, suggesting that LC8 stabilizes microtubules. We also show that LC8 knockdown or transfection with inhibitory peptides destabilizes microtubules and inhibits bipolar spindle assembly in HeLa cells. In addition, LC8 knockdown leads to the mitotic block in HeLa cells. Furthermore, molecular docking analysis using the crystal structures of tubulin and LC8 dimer indicated that the latter may bind at α-ß tubulin junction in a protofilament at sites distinct from the kinesin and dynein binding sites. Together, we provide the first evidence of a novel microtubule-associated protein-like function of LC8 that could explain its reported roles in cellular metastasis and differentiation.

Distance-Dependent Metal-Enhanced Intrinsic Fluorescence of Proteins Using Polyelectrolyte Layer-by-Layer Assembly and Aluminum Nanoparticles.

Previously reported studies indicate that aluminum nanostructured substrates can potentially find widespread use in metal-enhanced fluorescence (MEF) applications particularly in the UV or near-UV spectral region toward label-free detection of biomolecules. MEF largely depends on several factors, such as chemical nature, size, shape of the nanostructure and its distance from the fluorophore. A detailed understanding of the MEF and its distance-dependence are important for its potential application in biomedical sensing. Our goal is to utilize intrinsic protein fluorescence for label-free binding assays. This is made possible by the use of metallic nanostructures which provide localized excitation and enhanced fluorescence of UV fluorophores and will also provide a way to separate the surface-bound proteins from the bulk samples. We evaluated varied probe distances from plasmonic nanostructures by the well-established layer-by-layer (LbL) technique. The investigated proteins were adsorbed on different numbers of alternate layers of poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). Bovine serum albumin (BSA) was electrostatically attached to the positively charged PAH layer, and goat and rabbit IgG were attached to negatively charged PSS layer. We obtained a maximum of a ~ 9 fold increase in fluorescence intensity from BSA at a distance of ~9 nm from the Al nanostructured surface. Approximately 6- and 7- fold increases were observed from goat and rabbit IgG at a distance of ~8 nm, respectively. The minimum lifetimes were about 3-fold shorter than those on bare control quartz slides for all three proteins. The time-resolved intensity decays were analyzed with a lifetime distribution model to understand the distance effect on the metal-fluorophore interaction in detail. The present study indicates the distance dependence nature of metal-enhanced intrinsic fluorescence of proteins and potential of LbL assembly to control the metal-to-fluorophore distance in the UV wavelength region.

Distance-dependent intrinsic fluorescence of proteins on aluminum nanostructures.

In the past several years we have demonstrated the metal-enhanced fluorescence (MEF) and the significant changes in the photophysical properties of fluorophores in the presence of metallic nanostructures and nanoparticles using ensemble spectroscopic studies. In the represented study, we explored the distance effect on intrinsic fluorescence of proteins adsorbed on our layer-by-layer assembled metallic nanostructures. The study is expected to provide more information on the importance of positioning the proteins at a particular distance for enhanced fluorescence from metallic structures. For the present study, we considered using easy and inexpensive LbL technique to provide well-defined distance from metallic surface. The explored proteins were adsorbed on different numbers of alternate layers of poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). SA and BSA were electrostatically attached to the positively charged PAH layer. We obtained a maximum of ~11-fold and 9-fold increase in fluorescence intensity from SA and BSA, respectively. And also we observed ~3-fold decrease in BSA lifetime on metallic nanostructures than those on bare control quartz slides. This study reveals the distance dependence of protein fluorescence.

Dynein light chain 1 functions in somatic cyst cells regulate spermatogonial divisions in Drosophila.

Stem cell progeny often undergo transit amplifying divisions before differentiation. In Drosophila, a spermatogonial precursor divides four times within an enclosure formed by two somatic-origin cyst cells, before differentiating into spermatocytes. Although germline and cyst cell-intrinsic factors are known to regulate these divisions, the mechanistic details are unclear. Here, we show that loss of dynein-light-chain-1 (DDLC1/LC8) in the cyst cells eliminates bag-of-marbles (bam) expression in spermatogonia, causing gonial cell hyperplasia in Drosophila testis. The phenotype is dominantly enhanced by Dhc64C (cytoplasmic Dynein) and didum (Myosin V) loss-of-function alleles. Loss of DDLC1 or Myosin V in the cyst cells also affects their differentiation. Furthermore, cyst cell-specific loss of ddlc1 disrupts Armadillo, DE-cadherin and Integrin-ßPS localizations in the cyst. Together, these results suggest that Dynein and Myosin V activities, and independent DDLC1 functions in the cyst cells organize the somatic microenvironment that regulates spermatogonial proliferation and differentiation.

The use of aluminum nanostructures as platforms for metal enhanced fluorescence of the intrinsic emission of biomolecules in the ultra-violet.

We consider the possibility of using aluminum nanostructures for enhancing the intrinsic emission of biomolecules. We used the finite-difference time-domain (FDTD) method to calculate the effects of aluminum nanoparticles on nearby fluorophores that emit in the ultra-violet (UV). We find that the radiated power of UV fluorophores is significantly increased when they are in close proximity to aluminum nanostructures. We show that there will be increased localized excitation near aluminum particles at wavelengths used to excite intrinsic biomolecule emission. We also examine the effect of excited-state fluorophores on the near-field around the nanoparticles. Finally we present experimental evidence showing that a thin film of amino acids and nucleotides display enhanced emission when in close proximity to aluminum nanostructured surfaces. Our results suggest that biomolecules can be detected and identified using aluminum nanostructures that enhance their intrinsic emission. We hope this study will ignite interest in the broader scientific community to take advantage of the plasmonic properties of aluminum and the potential benefits of its interaction with biomolecules to generate momentum towards implementing fluorescence-based bioassays using their intrinsic emission.

Enhanced fluorescence of proteins and label-free bioassays using aluminum nanostructures.

We report the enhanced intrinsic fluorescence from several proteins in proximity to aluminum nanostructured surfaces. Intrinsic fluorescence in proteins is dominated by the tryptophan residues. Intensities and lifetimes of several proteins with different numbers of tryptophan residues assembled on the surfaces of quartz or aluminum nanostructured films were measured. Immobilized protein molecules on the surface of an aluminum nanostructured film resulted in a significant fluorescence intensity enhancement (up to 14-fold) and lifetime decrease (up to 6-fold) compared to the quartz substrates. These large spectroscopic changes allow design of label-free bioassays where detection of binding interactions between proteins can be observed in the presence of a bulk sample solution. Binding of streptavidin to the biotinylated aluminum surface was demonstrated in the presence of 100 microg/mL bovine serum albumin in the sample solution by measurements of tryptophan intensity and lifetime changes.

F-actin-based extensions of the head cyst cell adhere to the maturing spermatids to maintain them in a tight bundle and prevent their premature release in Drosophila testis.

BACKGROUND: In Drosophila, all the 64 clonally derived spermatocytes differentiate in syncytium inside two somatic-origin cyst cells. They elongate to form slender spermatids, which are individualized and then released into the seminal vesicle. During individualization, differentiating spermatids are organized in a tight bundle inside the cyst, which is expected to play an important role in sperm selection. However, actual significance of this process and its underlying mechanism are unclear. RESULTS: We show that dynamic F-actin-based processes extend from the head cyst cell at the start of individualization, filling the interstitial space at the rostral ends of the maturing spermatid bundle. In addition to actin, these structures contained lamin, beta-catenin, dynamin, myosin VI and several other filopodial components. Further, pharmacological and genetic analyses showed that cytoskeletal stability and dynamin function are essential for their maintenance. Disruption of these F-actin based processes was associated with spermatid bundle disassembly and premature sperm release inside the testis. CONCLUSION: Altogether, our data suggests that the head cyst cell adheres to the maturing spermatid heads through F-actin-based extensions, thus maintaining them in a tight bundle. This is likely to regulate mature sperm release into the seminal vesicle. Overall, this process bears resemblance to mammalian spermiation.