The role of mitochondria in sex- and age-specific gene expression in a species without sex chromosomes.

Mitochondria perform an array of functions, many of which involve interactions with gene products encoded by the nucleus. These mitochondrial functions, particularly those involving energy production, can be expected to differ between sexes and across ages. Here, we measured mitochondrial effects on sex- and age-specific gene expression in parental and reciprocal F1 hybrids between allopatric populations of Tigriopus californicus with over 20% mitochondrial DNA divergence. Because the species lacks sex chromosomes, sex-biased mitochondrial effects are not confounded by the effects of sex chromosomes. Results revealed pervasive sex differences in mitochondrial effects, including effects on energetics and aging involving nuclear interactions throughout the genome. Using single-individual RNA sequencing, sex differences were found to explain more than 80% of the variance in gene expression. Males had higher expression of mitochondrial genes and mitochondrially targeted proteins (MTPs) involved in oxidative phosphorylation (OXPHOS), while females had elevated expression of non-OXPHOS MTPs, indicating strongly sex-dimorphic energy metabolism at the whole organism level. Comparison of reciprocal F1 hybrids allowed insights into the nature of mito-nuclear interactions, showing both mitochondrial effects on nuclear expression, and nuclear effects on mitochondrial expression. While based on a small set of crosses, sex-specific increases in mitochondrial expression with age were associated with longer life. Network analyses identified nuclear components of strong mito-nuclear interactions and found them to be sexually dimorphic. These results highlight the profound impact of mitochondria and mito-nuclear interactions on sex- and age-specific gene expression.

The mRNA-capping enzyme localizes to stress granules in the cytoplasm and maintains cap homeostasis of target mRNAs.

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.

Not just binary: embracing the complexity of nuclear division dynamics.

Cell division presents a challenge for eukaryotic cells: how can chromosomes effectively segregate within the confines of a membranous nuclear compartment? Different organisms have evolved diverse solutions by modulating the degree of nuclear compartmentalization, ranging from complete nuclear envelope breakdown to complete maintenance of nuclear compartmentalization via nuclear envelope expansion. Many intermediate forms exist between these extremes, suggesting that nuclear dynamics during cell division are surprisingly plastic. In this review, we highlight the evolutionary diversity of nuclear divisions, focusing on two defining characteristics: (1) chromosome compartmentalization and (2) nucleocytoplasmic transport. Further, we highlight recent evidence that nuclear behavior during division can vary within different cellular contexts in the same organism. The variation observed within and between organisms underscores the dynamic evolution of nuclear divisions tailored to specific contexts and cellular requirements. In-depth investigation of diverse nuclear divisions will enhance our understanding of the nucleus, both in physiological and pathological states.

Axis formation in annual killifish: Nodal and ß-catenin regulate morphogenesis without Huluwa prepatterning.

Axis formation in fish and amphibians typically begins with a prepattern of maternal gene products. Annual killifish embryogenesis, however, challenges prepatterning models as blastomeres disperse and then aggregate to form the germ layers and body axes. We show that huluwa, a prepatterning factor thought to break symmetry by stabilizing ß-catenin, is truncated and inactive in Nothobranchius furzeri. Nuclear ß-catenin is not selectively stabilized on one side of the blastula but accumulates in cells forming the aggregate. Blocking ß-catenin activity or Nodal signaling disrupts aggregate formation and germ layer specification. Nodal signaling coordinates cell migration, establishing an early role for this signaling pathway. These results reveal a surprising departure from established mechanisms of axis formation: Huluwa-mediated prepatterning is dispensable, and ß-catenin and Nodal regulate morphogenesis.

Nucleoporin Nup358 drives the differentiation of myeloid-biased multipotent progenitors by modulating HDAC3 nuclear translocation.

Nucleoporins, the components of nuclear pore complexes (NPCs), can play cell type- and tissue-specific functions. Yet, the physiological roles and mechanisms of action for most NPC components have not yet been established. We report that Nup358, a nucleoporin linked to several myeloid disorders, is required for the developmental progression of early myeloid progenitors. We found that Nup358 ablation in mice results in the loss of myeloid-committed progenitors and mature myeloid cells and the accumulation of myeloid-primed multipotent progenitors (MPPs) in bone marrow. Accumulated MPPs in Nup358 knockout mice are greatly restricted to megakaryocyte/erythrocyte-biased MPP2, which fail to progress into committed myeloid progenitors. Mechanistically, we found that Nup358 is required for histone deacetylase 3 (HDAC3) nuclear import and function in MPP2 cells and established that this nucleoporin regulates HDAC3 nuclear translocation in a SUMOylation-independent manner. Our study identifies a critical function for Nup358 in myeloid-primed MPP2 differentiation and uncovers an unexpected role for NPCs in the early steps of myelopoiesis.

The UF-5000 Atyp.C parameter is an independent risk factor for bladder cancer.

Bladder carcinoma (BC) accounts for > 90% of all urothelial cancers. Pathological diagnosis through cytoscopic biopsy is the gold standard, whereas non-invasive diagnostic tools remain lacking. The "Atyp.C" parameter of the Sysmex UF-5000 urine particle analyzer represents the ratio of nucleus to cytoplasm and can be employed to detect urinary atypical cells. The present study examined the association between urinary Atyp.C values and BC risk. This two-center, retrospective case-control study identified clinical primary or newly recurrent BC (study period, 2022-2023; n = 473) cases together with controls with urinary tract infection randomly matched by age and sex (1:1). Urinary sediment differences were compared using non-parametric tests. The correlations between urinary Atyp.C levels and BC grade or infiltration were analyzed using Spearman's rank correlation. The BC risk factor odds ratio of Atyp.C was calculated using conditional logistic regression, and potential confounder effects were adjusted using stepwise logistic regression (LR). Primary risk factors were identified by stratified analysis according to pathological histological diagnosis. The mean value of urinary Atyp.C in BC cases (1.30 ± 3.12) was 8.7 times higher than that in the controls (0.15 ± 0.68; P < 0.001). Urinary Atyp.C values were positively correlated with BC pathological grade and invasion (r = 0.360, P < 0.001; r = 0.367, P < 0.001). Urinary Atyp.C was an independent risk factor for BC and closely related with BC pathological grade and invasion. Elevated urinary Atyp.C values was an independent risk factor for BC. Our findings support the use of Atyp.C as a marker that will potentially aid in the early diagnosis and long-term surveillance of new and recurrent BC cases.

The small tumor antigen of Merkel cell polyomavirus accomplishes cellular transformation by uniquely localizing to the nucleus despite the absence of a known nuclear localization signal.

BACKGROUND: Merkel Cell Carcinoma (MCC) is an aggressive skin cancer that is three times deadlier than melanoma. In 2008, it was found that 80% of MCC cases are caused by the genomic integration of a novel polyomavirus, Merkel Cell Polyomavirus (MCPyV), and the expression of its small and truncated large tumor antigens (ST and LT-t, respectively). MCPyV belongs to a family of human polyomaviruses; however, it is the only one with a clear association to cancer. METHODS: To investigate the role and mechanisms of various polyomavirus tumor antigens in cellular transformation, Rat-2 and 293A cells were transduced with pLENTI MCPyV LT-t, MCPyV ST, TSPyV ST, HPyV7 ST, or empty pLENTI and assessed through multiple transformation assays, and subcellular fractionations. One-way ANOVA tests were used to assess statistical significance. RESULTS: Soft agar, proliferation, doubling time, glucose uptake, and serum dependence assays confirmed ST to be the dominant transforming protein of MCPyV. Furthermore, it was found that MCPyV ST is uniquely transforming, as the ST antigens of other non-oncogenic human polyomaviruses such as Trichodysplasia Spinulosa-Associated Polyomavirus (TSPyV) and Human Polyomavirus 7 (HPyV7) were not transforming when similarly assessed. Identification of structural dissimilarities between transforming and non-transforming tumor antigens revealed that the uniquely transforming domain(s) of MCPyV ST are likely located within the structurally dissimilar loops of the MCPyV ST unique region. Of all known MCPyV ST cellular interactors, 62% are exclusively or transiently nuclear, suggesting that MCPyV ST localizes to the nucleus despite the absence of a canonical nuclear localization signal. Indeed, subcellular fractionations confirmed that MCPyV ST could achieve nuclear localization through a currently unknown, regulated mechanism independent of its small size, as HPyV7 and TSPyV ST proteins were incapable of nuclear translocation. Although nuclear localization was found to be important for several transforming properties of MCPyV ST, some properties were also performed by a cytoplasmic sequestered MCPyV ST, suggesting that MCPyV ST may perform different transforming functions in individual subcellular compartments. CONCLUSIONS: Together, these data further elucidate the unique differences between MCPyV ST and other polyomavirus ST proteins necessary to understand MCPyV as the only known human oncogenic polyomavirus.

Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality.

Signal recognition particles (SRPs) are essential for regulating intracellular protein transport and secretion. Patients with tumors with high SRP9 expression tend to have a poorer overall survival. However, to the best of our knowledge, no reports have described the relationship between SRP9 localization and prognosis in pancreatic cancer. Thus, the present study aimed to investigate this relationship. Immunohistochemical staining for SRP9 using excised specimens from pancreatic cancer surgery cases without preoperative chemotherapy or radiotherapy showed that SRP9 was preferentially expressed in the nucleus of the cancerous regions in some cases, which was hardly detected in other cases, indicating that SRP9 was transported to the nucleus in the former cases. To compare the prognosis of patients with SRP9 nuclear translocation, patients were divided into two groups: Those with a nuclear translocation rate of >50% and those with a nuclear translocation rate of ≤50%. The nuclear translocation rate of >50% group had a significantly better recurrence­free survival than the nuclear translocation rate of ≤50% group (P=0.037). Subsequent in vitro experiments were conducted; notably, the nuclear translocation rate of SRP9 was reduced under amino acid­deficient conditions, suggesting that multiple factors are involved in this phenomenon. To further study the function of SRP9 nuclear translocation, in vitro experiments were performed by introducing SRP9 splicing variants (v1 and v2) and their deletion mutants lacking C­terminal regions into MiaPaCa pancreatic cancer cells. The results demonstrated that both splicing variants showed nuclear translocation regardless of the C­terminal deletions, suggesting the role of the N­terminal regions. Given that SRP9 is an RNA­binding protein, the study of RNA immunoprecipitation revealed that signaling pathways involved in cancer progression and protein translation were downregulated in nuclear­translocated v1 and v2. Undoubtedly, further studies of the nuclear translocation of SRP9 will open an avenue to optimize the precise evaluation and therapeutic control of pancreatic cancer.

The bioenergetics of nucleocytoplasmic transport.

How nucleocytoplasmic transport (NCT) rates change due to cellular physiology-mediated fluctuations in GTP availability remains unclear. In this issue, Scott et al. (https://doi.org/10.1083/jcb.202308152) demonstrate that cell migration, spreading, and nucleocytoskeletal coupling impact GTP levels, thereby regulating NCT, RNA export, and protein synthesis.

MeCP2 binds to methylated DNA independently of phase separation and heterochromatin organisation.

Correlative evidence has suggested that the methyl-CpG-binding protein MeCP2 contributes to the formation of heterochromatin condensates via liquid-liquid phase separation. This interpretation has been reinforced by the observation that heterochromatin, DNA methylation and MeCP2 co-localise within prominent foci in mouse cells. The findings presented here revise this view. MeCP2 localisation is independent of heterochromatin as MeCP2 foci persist even when heterochromatin organisation is disrupted. Additionally, MeCP2 foci fail to show hallmarks of phase separation in live cells. Importantly, we find that mouse cellular models are highly atypical as MeCP2 distribution is diffuse in most mammalian species, including humans. Notably, MeCP2 foci are absent in Mus spretus which is a mouse subspecies lacking methylated satellite DNA repeats. We conclude that MeCP2 has no intrinsic tendency to form condensates and its localisation is independent of heterochromatin. Instead, the distribution of MeCP2 in the nucleus is primarily determined by global DNA methylation patterns.

Nuclear Softness Promotes the Metastatic Potential of Large-Nucleated Colorectal Cancer Cells via the ErbB4-Akt1-Lamin A/C Signaling Pathway.

Abnormal nuclear enlargement is a diagnostic and physical hallmark of malignant tumors. Large nuclei are positively associated with an increased risk of developing metastasis; however, a large nucleus is inevitably more resistant to cell migration due to its size. The present study demonstrated that the nuclear size of primary colorectal cancer (CRC) cells at an advanced stage was larger than cells at an early stage. In addition, the nuclei of CRC liver metastases were larger than those of the corresponding primary CRC tissues. CRC cells were sorted into large-nucleated cells (LNCs) and small-nucleated cells (SNCs). Purified LNCs exhibited greater constricted migratory and metastatic capacity than SNCs in vitro and in vivo. Mechanistically, ErbB4 was highly expressed in LNCs, which phosphorylated lamin A/C at serine 22 via the ErbB4-Akt1 signaling pathway. Furthermore, the level of phosphorylated lamin A/C was a negative determinant of nuclear stiffness. Taken together, CRC LNCs possessed greater constricted migratory and metastatic potential than SNCs due to ErbB4-Akt1-mediated lamin A/C phosphorylation and nuclear softening. These results may provide a potential treatment strategy for tumor metastasis by targeting nuclear stiffness in patients with cancer, particularly CRC.

CLK2 mediates IκBα-independent early termination of NF-κB activation by inducing cytoplasmic redistribution and degradation.

Activation of the NF-κB pathway is strictly regulated to prevent excessive inflammatory and immune responses. In a well-known negative feedback model, IκBα-dependent NF-κB termination is a delayed response pattern in the later stage of activation, and the mechanisms mediating the rapid termination of active NF-κB remain unclear. Here, we showed IκBα-independent rapid termination of nuclear NF-κB mediated by CLK2, which negatively regulated active NF-κB by phosphorylating the RelA/p65 subunit of NF-κB at Ser180 in the nucleus to limit its transcriptional activation through degradation and nuclear export. Depletion of CLK2 increased the production of inflammatory cytokines, reduced viral replication and increased the survival of the mice. Mechanistically, CLK2 phosphorylated RelA/p65 at Ser180 in the nucleus, leading to ubiquitin‒proteasome-mediated degradation and cytoplasmic redistribution. Importantly, a CLK2 inhibitor promoted cytokine production, reduced viral replication, and accelerated murine psoriasis. This study revealed an IκBα-independent mechanism of early-stage termination of NF-κB in which phosphorylated Ser180 RelA/p65 turned off posttranslational modifications associated with transcriptional activation, ultimately resulting in the degradation and nuclear export of RelA/p65 to inhibit excessive inflammatory activation. Our findings showed that the phosphorylation of RelA/p65 at Ser180 in the nucleus inhibits early-stage NF-κB activation, thereby mediating the negative regulation of NF-κB.

Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind.

Developmental programming involves the accurate conversion of signalling levels and dynamics to transcriptional outputs. The transcriptional relay in the Notch pathway relies on nuclear complexes containing the co-activator Mastermind (Mam). By tracking these complexes in real time, we reveal that they promote the formation of a dynamic transcription hub in Notch ON nuclei which concentrates key factors including the Mediator CDK module. The composition of the hub is labile and persists after Notch withdrawal conferring a memory that enables rapid reformation. Surprisingly, only a third of Notch ON hubs progress to a state with nascent transcription, which correlates with polymerase II and core Mediator recruitment. This probability is increased by a second signal. The discovery that target-gene transcription is probabilistic has far-reaching implications because it implies that stochastic differences in Notch pathway output can arise downstream of receptor activation.

To correctly give rise to future tissues, cells in an embryo must receive and respond to the right signals, at the right time, in the right way. This involves genes being switched on quickly, with cells often ensuring that a range of molecular actors physically come together at 'transcription hubs' in the nucleus ­ the compartment that houses genetic information. These hubs are thought to foster a microenvironment that facilitates the assembly of the machinery that will activate and copy the required genes into messenger RNA molecules. The resulting 'mRNAs' act as templates for producing the corresponding proteins, allowing cells to adequately respond to signals. For example, the activation at the cell surface of a molecule called Notch triggers a series of events that lead to important developmental genes being transcribed within minutes. This process involves a dedicated group of proteins, known as Notch nuclear complexes, quickly getting together in the nucleus and interacting with the transcriptional machinery. How they do this efficiently at the right gene locations is, however, still poorly understood. In particular, it remained unclear whether Notch nuclear complexes participate in the formation of transcription hubs, as well as how these influence mRNA production and the way cells 'remember' having been exposed to Notch activity. To investigate these questions, DeHaro-Arbona et al. genetically engineered fruit flies so that their Notch nuclear complexes and Notch target genes both carried visible tags that could be tracked in living cells in real time. Microscopy imaging of fly tissues revealed that, due to their characteristics, Notch complexes clustered with the transcription machinery and formed transcription hubs near their target genes. All cells exposed to Notch exhibited these hubs, but only a third produced the mRNAs associated with Notch target genes; adding a second signal (an insect hormone) significantly increased the proportion. This illustrates how 'chance' and collaboration influence the way the organism responds to Notch signalling. Finally, the experiments revealed that the hubs persisted for at least a day after removing the Notch signal. This 'molecular memory' led to cells responding faster when presented with Notch activity again. The work by DeHaro-Arbona sheds light on how individual cells respond to Notch signalling, and the factors that influence the activation of its target genes. This knowledge may prove useful when trying to better understand diseases in which this pathway is implicated, such as cancer.

Vaccinia virus subverts xenophagy through phosphorylation and nuclear targeting of p62.

Autophagy is an essential degradation program required for cell homeostasis. Among its functions is the engulfment and destruction of cytosolic pathogens, termed xenophagy. Not surprisingly, many pathogens use various strategies to circumvent or co-opt autophagic degradation. For poxviruses, it is known that infection activates autophagy, which however is not required for successful replication. Even though these complex viruses replicate exclusively in the cytoplasm, autophagy-mediated control of poxvirus infection has not been extensively explored. Using the prototypic poxvirus, vaccinia virus (VACV), we show that overexpression of the xenophagy receptors p62, NDP52, and Tax1Bp1 restricts poxvirus infection. While NDP52 and Tax1Bp1 were degraded, p62 initially targeted cytoplasmic virions before being shunted to the nucleus. Nuclear translocation of p62 was dependent upon p62 NLS2 and correlated with VACV kinase mediated phosphorylation of p62 T269/S272. This suggests that VACV targets p62 during the early stages of infection to avoid destruction and further implies that poxviruses exhibit multi-layered control of autophagy to facilitate cytoplasmic replication.

p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol.

The AAA+-ATPase valosin-containing protein (VCP; also called p97 or Cdc48), a major protein unfolding machinery with a variety of essential functions, localizes to different subcellular compartments where it has different functions. However, the processes regulating the distribution of VCP between the cytosol and nucleus are not understood. Here, we identified p37 (also called UBXN2B) as a major factor regulating VCP nucleocytoplasmic shuttling. p37-dependent VCP localization was crucial for local cytosolic VCP functions, such as autophagy, and nuclear functions in DNA damage repair. Mutations in VCP causing multisystem proteinopathy enhanced its association with p37, leading to decreased nuclear localization of VCP, which enhanced susceptibility to DNA damage accumulation. Both VCP localization and DNA damage susceptibility in cells with such mutations were normalized by lowering p37 levels. Thus, we uncovered a mechanism by which VCP nucleocytoplasmic distribution is fine-tuned, providing a means for VCP to respond appropriately to local needs.

Nuclear sequences of mitochondrial origin in domestic yak.

Mitochondrial DNA sequences are frequently transferred into the nuclear genome, generating nuclear mitochondrial DNA sequences (NUMTs). Here, we analysed, for the first time, NUMTs in the domestic yak genome. We obtained 499 alignment matches covering 340.2 kbp of the yak nuclear genome. After a merging step, we identified 167 NUMT regions with a total length of ~ 503 kbp, representing 0.02% of the nuclear genome. We discovered copies of all mitochondrial regions and found that most NUMT regions are intergenic or intronic and mostly untranscribed. 98 different NUMT regions from domestic yak showed high homology with cow and/or wild yak genomes, suggesting selection or hybridization between domestic/wild yak and cow. To rule out the possibility that the identified NUMTs could be artifacts of the domestic yak genome assembly, we validated experimentally five NUMT regions by PCR amplification. As NUMT regions show high similarity to the mitochondrial genome can potentially pose a risk to domestic yak DNA mitochondrial studies, special care is therefore needed to select primers for PCR amplification of mitochondrial DNA sequences.

Deep sequencing of Phox2a nuclei reveals five classes of anterolateral system neurons.

The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch, and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here, we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify three clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 and ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons.

Single nucleus and spatial transcriptomic profiling of healthy human hamstring tendon.

The molecular and cellular basis of health in human tendons remains poorly understood. Among human tendons, hamstring tendon has markedly low pathology and can provide a prototypic healthy tendon reference. The aim of this study was to determine the transcriptomes and location of all cell types in healthy hamstring tendon. Using single nucleus RNA sequencing, we profiled the transcriptomes of 10 533 nuclei from four healthy donors and identified 12 distinct cell types. We confirmed the presence of two fibroblast cell types, endothelial cells, mural cells, and immune cells, and identified cell types previously unreported in tendons, including different skeletal muscle cell types, satellite cells, adipocytes, and undefined nervous system cells. The location of these cell types within tendon was defined using spatial transcriptomics and imaging, and potential transcriptional networks and cell-cell interactions were analyzed. We demonstrate that fibroblasts have the highest number of potential cell-cell interactions in our dataset, are present throughout the tendon, and play an important role in the production and organization of extracellular matrix, thus confirming their role as key regulators of hamstring tendon homeostasis. Overall, our findings underscore the complexity of the cellular networks that underpin healthy human tendon function and the central role of fibroblasts as key regulators of hamstring tendon tissue homeostasis.

Imaging HIV-1 Nuclear Import, Uncoating, and Proviral Transcription.

Live-cell imaging has become a powerful tool for dissecting the behavior of viral complexes during HIV-1 infection with high temporal and spatial resolution. Very few HIV-1 particles in a viral population are infectious and successfully complete replication (~1/50). Single-particle live-cell imaging enables the study of these rare infectious viral particles, which cannot be accomplished in biochemical assays that measure the average property of the entire viral population, most of which are not infectious. The timing and location of many events in the early stage of the HIV-1 life cycle, including nuclear import, uncoating, and integration, have only recently been elucidated. Live-cell imaging also provides a valuable approach to study interactions of viral and host factors in distinct cellular compartments and at specific stages of viral replication. Successful live-cell imaging experiments require careful consideration of the fluorescent labeling method used and avoid or minimize its potential impact on normal viral replication and produce misleading results. Ideally, it is beneficial to utilize multiple virus labeling strategies and compare the results to ensure that the virion labeling did not adversely influence the viral replication step that is under investigation. Another potential benefit of using different labeling strategies is that they can provide information about the state of the viral complexes. Here, we describe our methods that utilize multiple fluorescent protein labeling approaches to visualize and quantify important events in the HIV-1 life cycle, including docking HIV-1 particles with the nuclear envelope (NE) and their nuclear import, uncoating, and proviral transcription.