DNAL1 Human

What is DNAL1 and what is its primary function in human cells?

DNAL1 (Dynein Axonemal Light Chain 1) is a protein that functions as a light intermediate chain (LIC) subunit of inner dynein arms (IDAs) in mammals. It plays a crucial role in ciliary and flagellar movement by participating in the hydrolysis of ATP that drives these motile structures. DNAL1 is the human ortholog of the Chlamydomonas axonemal dynein light chain 1 (LC1), which is a component of outer dynein arms (ODAs) responsible for ATP-dependent ciliary movement . The protein contains highly conserved domains, particularly the leucine-rich-repeat (LRR) consensus domain that is completely conserved across species, indicating its functional importance .

In which human tissues is DNAL1 predominantly expressed?

DNAL1 is predominantly expressed in tissues that contain motile cilia or flagella. These include the respiratory epithelium (trachea, bronchi, and lungs), brain ependymal cells (which line the ventricles and central canal), and male reproductive tissues, particularly in developing and mature spermatozoa. This tissue-specific expression pattern aligns with DNAL1's functional role in ciliary and flagellar motility, which is essential for mucus clearance in airways, cerebrospinal fluid movement in the brain, and sperm motility in reproduction .

How many isoforms of DNAL1 exist in humans and how do they differ?

At least two isoforms of DNAL1 have been identified in humans . While the search results don't provide specific details about the structural or functional differences between these isoforms, this variation may reflect tissue-specific requirements or developmental regulation. Researchers investigating DNAL1 should account for these isoforms in experimental design, particularly when developing antibodies, designing PCR primers, or conducting expression studies, as different isoforms may have distinct functions or expression patterns in various tissues.

What is the relationship between DNAL1 mutations and primary ciliary dyskinesia (PCD)?

DNAL1 has been identified as a causative gene for primary ciliary dyskinesia (PCD), a genetically heterogeneous disorder characterized by chronic infections of the upper and lower airways, randomization of left-right body symmetry, and reduced fertility. Homozygous mutations in DNAL1 can impair ciliary motility, leading to the clinical manifestations of PCD. The protein plays a crucial role in ciliary structure and function, particularly through its interaction with DNAH5, a protein that when mutated is known to cause PCD. DNAL1 likely serves a regulatory function for DNAH5 activity in the outer dynein arms of respiratory cilia and ependymal cilia .

How do DNAL1 mutations affect male fertility?

Mutations in DNAL1 have been directly linked to male infertility, specifically asthenospermia (ASZ), characterized by severely reduced sperm motility. A homozygous frameshift mutation in DNAL1 [c.663_666del (p.Glu221fs)] identified in an ASZ patient resulted in complete loss of the DNAL1 protein in sperm. This mutation led to disruptions in the dynein arm structure and affected the fibrous sheath (FS) of the sperm flagellum. DNAL1 deficiency causes asymmetries in the fibrous sheath and disrupts the transport and assembly of FS proteins, particularly AKAP3 and AKAP4, during flagellogenesis. The combined effect of inner dynein arms injury and asymmetric FS-driven tail rigid structure alteration leads to flagellum immotility and consequently male infertility .

What methodological approaches are used to diagnose DNAL1-related disorders in clinical settings?

Diagnosing DNAL1-related disorders typically involves a multi-faceted approach:

• Genetic Analysis: Whole-exome sequencing followed by bioinformatic analysis is the primary method to identify mutations in DNAL1. This involves DNA extraction, whole exome enrichment, and high-throughput sequencing using platforms such as HiSeq X-TEN or NovaSeq 6000. The sequencing data is then processed using standard assembly tools (e.g., Burrows-Wheeler Aligner), calling (Genome Analysis Toolkit), and annotation (ANNOVAR) .

• Validation: Sanger sequencing is performed to validate candidate mutations identified through whole-exome sequencing and to determine their origins (e.g., parental transmission patterns) .

• Protein Analysis: Immunological techniques using antibodies against DNAL1 can assess protein expression in affected tissues such as sperm samples .

• Phenotypic Assessment: Clinical evaluation including respiratory function tests, semen analysis for sperm motility, and assessment of left-right asymmetry may be conducted to correlate genetic findings with clinical manifestations .

What proteins are known to interact with DNAL1 in human cells?

DNAL1 interacts with several key proteins in human cells:

• DNAH5: DNAL1 interacts with DNAH5, a dynein heavy chain protein. This interaction suggests that DNAL1 serves a regulatory function for DNAH5 activity in outer dynein arms of sperm flagella, respiratory cilia, and ependymal cilia .

• DNAH1 and DNAH7: Loss of DNAL1 in both human patients and mouse models results in the loss of DNAH1 and DNAH7 (dynein heavy chain proteins) in sperm, indicating direct or indirect interactions between these proteins .

• Cytoplasmic Dynein Complex Proteins: Immunoprecipitation studies revealed that DNAL1 might interact with cytoplasmic dynein complex proteins in the testes, suggesting a role beyond axonemal dynein function .

• AKAP3 and AKAP4: DNAL1 appears to influence the transport and assembly of fibrous sheath proteins, especially AKAP3 and AKAP4, during flagellogenesis, suggesting functional interactions with these proteins .

These interactions highlight DNAL1's complex role in both axonemal structure formation and regulatory functions during spermatogenesis and ciliogenesis.

What is the role of DNAL1 in axonemal dynein arm assembly?

DNAL1 plays a critical role in the assembly and function of axonemal dynein arms, particularly inner dynein arms (IDAs) in mammals:

• IDA Component: DNAL1 functions as a light intermediate chain (LIC) subunit of inner dynein arms in mammals .

• Dynein Arm Integrity: Loss of DNAL1 results in the loss of specific dynein heavy chains (DNAH1 and DNAH7) rather than others (DNAH10), demonstrating that DNAL1 is required for the assembly or stability of a subset of inner dynein arm components .

• Subspecies Specificity: DNAL1 appears to be a LIC protein of a partial IDA subspecies, indicating its role in specific dynein arm substructures .

• Non-classical Function: Beyond its structural role in IDAs, DNAL1 may possess a non-classical molecular function in regulating the cytoplasmic dynein complex that assembles flagella, suggesting a role in trafficking components needed for axonemal assembly .

• Evolutionary Conservation: As the ortholog of the Chlamydomonas p28 protein (axonemal dynein light chain 1), DNAL1's role in dynein arm assembly is evolutionarily conserved, suggesting fundamental importance in ciliary and flagellar motility across species .

What are the recommended approaches for creating and validating DNAL1 knockout models?

Based on the successful development of Dnali1-knockout mice described in the search results, the following approaches are recommended:

• CRISPR/Cas9 Gene Editing:

  • Design sgRNAs targeting specific exons (e.g., exons 3-5 of Dnali1 as used in the referenced study)

  • Synthesize sgRNAs using appropriate primers (e.g., 5'-TGCATAAGACTATTAGGTGG AGG-3' and 5'-GTCATGGGAGTCCTCTCGGC AGG-3')

  • Linearize Cas9 and sgRNA plasmids with appropriate restriction enzymes (AgeI and DraI were used in the referenced study)

  • Purify the plasmids using PCR purification kits

  • Generate Cas9 mRNA using transcription kits (e.g., MESSAGE mMACHINE T7 Ultra Kit)

  • Generate purified sgRNA using appropriate kits (e.g., MEGA Shortscript and Clear Kit)

• Zygote Microinjection:

  • Mate superovulated wild-type females with males to produce zygotes

  • Microinject Cas9 mRNA and sgRNAs into zygotes

  • Transfer injected zygotes to pseudopregnant females

• Validation and Background Clearing:

  • Genotype founders using PCR to confirm gene editing

  • Mate edited founders with wild-type mice for multiple generations (at least three) to minimize off-target effects

  • Validate offspring genotypes using PCR with appropriate primers (e.g., F: 5'-CATGGGGTCTCAGTATGGTTGTAT-3'; R1: 5'- AGTGAATTCTGTGCTGGAGGAATA-3'; R2: 5'-TAATCCCTTCTCTTTCCCATCTGGT-3')

• Phenotypic Characterization:

  • Assess fertility and sperm motility in male knockout mice

  • Analyze sperm flagellar ultrastructure using electron microscopy

  • Perform immunofluorescence to assess protein expression and localization

  • Compare phenotypes with human patients carrying similar mutations to validate the model

What experimental design considerations are critical when studying DNAL1 in human fertility research?

When designing experiments to study DNAL1 in human fertility research, several critical considerations should be addressed:

• Patient Selection and Controls:

  • Carefully screen idiopathic asthenospermia patients, excluding those with abnormalities in somatic chromosome karyotype, genomic azoospermia factor deletions, serum sex hormone levels, and scrotal ultrasonography

  • Include appropriate control groups matched for age, ethnicity, and other relevant factors

  • Consider consanguinity factors, as demonstrated in the study involving a Chinese consanguineous family

• Ethical Considerations:

  • Obtain proper ethical approval from institutional review boards

  • Secure documented informed consent from all subjects prior to study initiation

  • Ensure compliance with the Declaration of Helsinki and relevant institutional ethical guidelines

• Comprehensive Genetic Analysis:

  • Perform whole-exome sequencing with appropriate bioinformatic analysis

  • Validate candidate mutations using Sanger sequencing

  • Consider family segregation analysis to confirm inheritance patterns

• Protein Expression Analysis:

  • Use appropriate antibodies for detecting DNAL1 and interacting proteins

  • Include relevant controls such as DNAH1, DNAH7, DNAH10, AKAP3, and AKAP4

  • Assess both presence/absence and localization of proteins

• Functional Assessments:

  • Analyze sperm motility parameters using computer-assisted sperm analysis

  • Examine flagellar ultrastructure using electron microscopy

  • Consider immunoprecipitation studies to identify protein interactions

• Translational Applications:

  • Evaluate assisted reproductive technology outcomes (e.g., ICSI success rates) in patients with DNAL1 mutations

  • Consider genetic counseling implications for affected families

How can researchers avoid batch effects and experimental design flaws when studying DNAL1 gene variants?

Researchers studying DNAL1 gene variants should implement rigorous experimental design practices to avoid batch effects and other common flaws:

• Proper Randomization:

  • Randomize all aspects of data collection and experimental order (including plating) with respect to phenotypes of interest

  • Avoid systematic biases such as processing all cases in one batch and controls in another, which was a problem in many studies, including the Wellcome Trust Case Control Consortium

• Sample Processing Controls:

  • Process cases and controls together in the same batches

  • Distribute samples across batches based on key variables (e.g., phenotype, sex, age) to minimize systematic biases

  • Include technical replicates across batches to detect batch effects

• Data Analysis Considerations:

  • Include batch as a covariate in statistical analyses

  • Use appropriate statistical methods to account for potential confounding variables

  • Apply batch correction algorithms when appropriate, but be cautious not to remove true biological signals

• Mega-Analysis Precautions:

  • Exercise caution when combining multiple experiments to increase statistical power, as confounding can worsen when poorly randomized experiments are combined

  • Verify compatibility of data collection methods before merging datasets

  • Apply appropriate meta-analysis techniques rather than simply pooling data

• Quality Control:

  • Implement rigorous quality control metrics throughout the experimental workflow

  • Document all steps in the experimental process, including any deviations from protocols

  • Validate findings using alternative methodologies or independent sample sets

These practices are particularly important given that approximately 95% of genetic studies analyzed by Golden Helix had major problems with experimental design, leading to spurious associations that could not be distinguished from real genetic associations .

What are the current contradictions or knowledge gaps in understanding DNAL1's role in primary ciliary dyskinesia versus male infertility?

Several important contradictions and knowledge gaps exist in our understanding of DNAL1's role in different clinical manifestations:

• Structural Role Discrepancies:

  • DNAL1 has been described as both a component of inner dynein arms (IDAs) in some studies and as an outer dynein arm (ODA) component in others , suggesting either dual functionality or context-dependent roles that require further clarification.

  • The precise subspecies of dynein arms that incorporate DNAL1 remains incompletely characterized, as loss of DNAL1 affects some dynein heavy chains (DNAH1, DNAH7) but not others (DNAH10) .

• Phenotypic Spectrum:

  • While some studies emphasize DNAL1's role primarily in male infertility (asthenospermia) , others highlight its importance in primary ciliary dyskinesia, which has broader clinical manifestations including respiratory symptoms and left-right asymmetry defects .

  • The factors determining whether DNAL1 mutations manifest primarily as isolated male infertility versus syndromic PCD remain unclear.

• Molecular Mechanism Gaps:

  • The non-classical function of DNAL1 in regulating cytoplasmic dynein complexes that assemble flagella needs further investigation, as this suggests roles beyond structural components of axonemal dynein arms .

  • The specific interactions between DNAL1 and fibrous sheath proteins (AKAP3, AKAP4) during flagellogenesis require more detailed molecular characterization .

• HIV Dependency Factor Role:

  • DNAL1 has been identified as an HIV dependency factor (HDF) , but the mechanism underlying this function and its relationship to DNAL1's ciliary/flagellar roles remains unexplored.

  • This unexpected function suggests DNAL1 may have broader cellular roles than currently appreciated.

Future research should address these contradictions through comprehensive structural biology approaches, broader phenotypic characterization of patients with DNAL1 mutations, and detailed molecular interaction studies.

How can advanced imaging techniques enhance our understanding of DNAL1 function in human cells?

Advanced imaging techniques offer powerful approaches to elucidate DNAL1 function:

• Super-Resolution Microscopy:

  • Techniques such as structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), and single-molecule localization microscopy (PALM/STORM) can resolve the nanoscale organization of DNAL1 within dynein arms.

  • These approaches can help resolve contradictions regarding DNAL1's localization in inner versus outer dynein arms and visualize its interactions with other dynein components.

• Cryo-Electron Microscopy (Cryo-EM):

  • High-resolution structural analysis of dynein arm complexes containing DNAL1 can provide insights into how DNAL1 contributes to dynein arm assembly and function.

  • Comparing structures with and without DNAL1 can reveal conformational changes and functional mechanisms.

• Live Cell Imaging:

  • Fluorescently tagged DNAL1 combined with high-speed confocal or light sheet microscopy can track DNAL1's dynamics during ciliogenesis and flagellogenesis.

  • FRAP (Fluorescence Recovery After Photobleaching) can assess DNAL1 turnover rates in cilia and flagella.

• Correlative Light and Electron Microscopy (CLEM):

  • CLEM can bridge the gap between light microscopy localization of DNAL1 and ultrastructural analysis of its environment within dynein arms.

  • This approach is particularly valuable for understanding DNAL1's role in the fibrous sheath asymmetries observed in knockout models .

• Expansion Microscopy:

  • Physical expansion of preserved cellular structures combined with standard fluorescence microscopy can achieve super-resolution-like imaging of DNAL1 localization.

  • This technique is particularly useful for visualizing DNAL1's distribution throughout the length of cilia and flagella.

These advanced imaging approaches, combined with genetic models and biochemical analyses, will provide more comprehensive insights into DNAL1's multifaceted functions in ciliary and flagellar biology.

What are the most promising therapeutic approaches for addressing DNAL1-related disorders?

While direct therapeutic interventions for DNAL1-related disorders remain limited, several promising approaches are emerging:

• Assisted Reproductive Technologies:

  • Intracytoplasmic sperm injection (ICSI) has been shown to effectively resolve infertility in patients with DNAL1 mutations .

  • This approach bypasses the motility defects caused by DNAL1 deficiency by directly injecting sperm into oocytes.

• Gene Therapy Approaches:

  • Adeno-associated virus (AAV)-mediated gene delivery could potentially restore DNAL1 expression in affected tissues.

  • For respiratory manifestations of PCD, inhaled gene therapy targeting airway epithelial cells could improve mucociliary clearance.

  • The challenge lies in achieving sustained expression in the appropriate cell types and ensuring proper protein integration into existing structures.

• Pharmacological Modulation of Downstream Pathways:

  • Identifying and targeting pathways downstream of DNAL1 dysfunction may provide symptomatic relief.

  • For respiratory symptoms, mucolytics and targeted anti-inflammatory agents may help manage airway manifestations of PCD.

• CRISPR-Based Therapeutic Strategies:

  • Advances in CRISPR technology could eventually enable correction of DNAL1 mutations in affected tissues or germline cells.

  • This approach faces significant technical and ethical challenges but represents a potential future direction.

• Personalized Medicine Approaches:

  • Comprehensive genotype-phenotype correlation studies could help predict disease severity and progression.

  • Such information would enable tailored management strategies for individual patients based on their specific DNAL1 mutations and expected clinical course.

The development of these therapeutic approaches requires continued basic research into DNAL1's functions and careful clinical studies to assess efficacy and safety in patients with DNAL1-related disorders.