Recombinant Mouse SUN domain-containing protein 5 (Sun5)

What are the primary biological functions of Sun5 in normal physiology?

Sun5 plays multiple critical roles in cellular function:

• Spermatogenesis: Sun5 is essential for proper sperm head-to-tail connection. Knockout studies demonstrate that Sun5 deletion causes decapitated sperm in the epididymis, resulting in acephalic spermatozoa syndrome .

• Nuclear-cytoskeletal linking: Sun5 forms part of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex by interacting with Nesprin3. This complex is crucial for maintaining the physical connection between the sperm head and tail .

• Cell division: Sun5 was first identified as a nuclear envelope protein involved in spermatocyte division and nuclear migration during meiosis .

• Structural integrity: During spermiogenesis, Sun5 helps maintain the proper positioning of the centrosome (the originator of the flagellum) relative to the nucleus .

What are the optimal conditions for handling recombinant mouse Sun5 protein?

For research using recombinant mouse Sun5 protein, the following handling protocols are recommended:

Storage conditions:

• Store lyophilized protein at -20°C/-80°C upon receipt

• Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (recommended 50%)

• Aliquot for long-term storage at -20°C/-80°C

Buffer composition:

• Tris/PBS-based buffer with 6% Trehalose, pH 8.0

What expression systems are most effective for producing recombinant Sun5?

Based on research protocols, E. coli has been successfully used as an expression system for recombinant mouse Sun5 protein . For mammalian expression, lentiviral systems have proven effective:

Lentiviral expression protocol:

• Clone full-length coding sequence of Sun5 with appropriate tag (e.g., Flag tag) into a lentiviral vector such as pLVX-IRES-Puro

• Package recombinant and control plasmids into lentivirus with viral titers >10^8 TU

• Co-culture lentiviruses with target cells for 12-18 hours in the presence of polybrene

• After 72 hours post-transfection, maintain stably transfected cells in medium containing 2 μg/mL puromycin

• Confirm Sun5 expression using Western blotting

How does Sun5 interact with the LINC complex components and what methods best capture these interactions?

Sun5 forms a critical component of the LINC complex primarily through its interaction with Nesprin3. This interaction is essential for connecting the sperm head and tail.

Interaction mechanism:
Sun5 localizes to the nuclear membrane where it interacts with Nesprin3. During spermatogenesis, Nesprin3 is located both posterior and anterior to the nucleus. In Sun5 knockout mice, Nesprin3 loses its localization at the implantation fossa of the posterior region, causing the centrosome to become distant from the nucleus and leading to head-tail separation .

Recommended methods for studying these interactions:

• Co-Immunoprecipitation (Co-IP) protocol:

  • Extract total protein from Sun5 stable overexpression cell line

  • Lyse with IP lysis solution containing phosphatase inhibitors

  • Pre-bond antibodies to magnetic beads (4°C, 6-8 hours)

  • Incubate total protein with the magnetic bead/antibodies complexes (4°C, overnight with rotation)

  • Perform Western blotting on the purified protein

• Immunofluorescence assays: To visualize the co-localization of Sun5 with Nesprin3 and other LINC complex components

• Ultrastructural imaging: To observe the head-tail coupling apparatus (HTCA) and the relationship between the centrosome and nucleus during spermatid development

What is the role of Sun5 in cancer progression and what are its downstream signaling mechanisms?

Recent research has revealed that Sun5 plays a significant role in colorectal cancer (CRC) progression through multiple mechanisms:

Expression profile:
Sun5 is highly expressed in CRC tissues and cells compared to adjacent normal tissues, suggesting a potential oncogenic role .

Functional effects:

• Promotes cell proliferation and migration in vitro

• Accelerates tumor formation in vivo as demonstrated in xenograft transplantation experiments

Signaling pathway interactions:

• ERK pathway regulation: Sun5 overexpression upregulates phosphorylated ERK1/2 (pERK1/2), while Sun5 knockdown reduces pERK1/2 levels

• Mechanism of ERK pathway regulation:

  • Sun5 regulates the ERK pathway via Nesprin2 mediation

  • Sun5 promotes nuclear translocation of pERK1/2 by interacting with Nup93

• Drug resistance implications: Overexpression of Sun5 partially reverses the inhibitory effects of PD0325901 (an ERK pathway inhibitor) on cell proliferation and migration, suggesting Sun5 overexpression may contribute to drug resistance in cancer treatment

Experimental approaches to study Sun5 in cancer:

• RNA-Seq analysis for identification of differentially expressed genes in control versus Sun5-overexpression cells

• Western blotting to assess protein expression levels and pathway activation

• In vitro proliferation and migration assays

• Xenograft models for in vivo validation

What are the molecular consequences of Sun5 knockout or mutation?

Sun5 knockout or mutation leads to specific phenotypic and molecular consequences:

In reproductive biology:

• Decapitated sperm in the epididymis, resulting in acephalic spermatozoa syndrome

• Disruption of the sperm head-to-tail connection due to damage of the Sun5/Nesprin3 complex

• Malformation of the head-tail coupling apparatus (HTCA)

Molecular mechanism of sperm defects:

• During normal spermatid elongation (steps 9-10), the HTCA and centrosome remain properly positioned near the nucleus

• In Sun5 knockout mice, the HTCA and centrosome become distant from the nucleus during these critical steps

• When the manchette disappears at steps 13-14, the head and tail separate completely

• Nesprin3 mislocalization: Without Sun5, Nesprin3 fails to localize properly at the implantation fossa of the posterior region of the nucleus

Experimental models:
Sun5 knockout mice (Sun5−/−) serve as valuable models for studying acephalic spermatozoa syndrome, which affects approximately 33-47% of human patients with this condition due to SUN5 mutations .

What are the most effective antibodies and detection methods for studying Sun5?

Based on published research protocols, the following approaches are recommended:

Antibodies successfully used in Sun5 research:

• Anti-SUN5 antibody (Thermo Fisher Scientific, 1:1000 dilution for Western blotting)

Detection protocols:

• Western blotting protocol:

  • Lyse tissues in RIPA buffer with PMSF on ice after grinding with liquid nitrogen

  • For cells: collect, wash with PBS, and lyse in RIPA with protease inhibitor cocktail

  • Perform electrophoresis and transfer proteins to PVDF membranes

  • Block with 5% non-fat milk (room temperature, 2 hours)

  • Incubate with primary antibody (4°C, overnight)

  • Wash with TBST three times

  • Incubate with HRP-coupled secondary antibodies (room temperature, 1 hour)

  • Visualize using ECL kit and document with an appropriate imaging system

• Immunofluorescence for localization studies:
  Particularly effective for visualizing Sun5's position relative to other nuclear envelope and LINC complex components

How can researchers effectively generate stable Sun5-expressing or Sun5-knockdown cell lines?

Overexpression protocol:

• Clone the full-length coding sequence of Sun5 with a tag (e.g., Flag tag) into a lentiviral vector (e.g., pLVX-IRES-Puro)

• Package recombinant and control plasmids into lentivirus (viral titers >10^8 TU)

• Co-culture lentiviruses with target cells for 12-18 hours with polybrene

• After 72 hours post-transfection, maintain stably transfected cells in medium with 2 μg/mL puromycin

• Confirm Sun5 expression by Western blotting

Knockdown protocol:

• Design short hairpin RNA (shRNA) sequences targeting the Sun5 gene

• Insert into an appropriate vector (e.g., pLV3ltr-ZsGreen-Puro-U6)

• Package into lentivirus

• Infect target cells according to manufacturer's recommendations

• After 72 hours, evaluate infection efficiency using fluorescence microscopy (if using a vector with fluorescent marker)

• Select stable cells using 2 μg/mL puromycin

• Verify knockdown efficiency by Western blotting

What are the emerging areas of Sun5 research beyond reproductive biology?

While Sun5 was initially characterized for its role in spermatogenesis, recent findings suggest broader implications:

• Cancer biology: The discovery of Sun5's role in colorectal cancer opens new avenues for cancer research. Sun5's ability to promote cell proliferation, migration, and modulate the ERK pathway suggests potential roles in other cancer types .

• Nuclear envelope dynamics: As a member of the SUN protein family involved in the LINC complex, Sun5 may have undiscovered functions in general nuclear envelope biology, nuclear positioning, and mechanotransduction.

• Signaling pathway modulation: Sun5's ability to affect ERK pathway activation suggests potential involvement in other signaling pathways relevant to development, homeostasis, and disease.

• Therapeutic targeting: Understanding Sun5's role in drug resistance to ERK pathway inhibitors could lead to novel combination therapies for cancer treatment .

How might Sun5 dysfunction contribute to human pathologies beyond acephalic spermatozoa syndrome?

Given the emerging roles of Sun5 in cellular functions:

• Cancer progression: Elevated Sun5 expression in colorectal cancer suggests it may serve as a biomarker or therapeutic target in multiple cancer types .

• Drug resistance mechanisms: Sun5's ability to partially reverse ERK inhibitor effects suggests it may contribute to treatment resistance in cancers .

• Nuclear envelope disorders: Given its role in the LINC complex, Sun5 dysfunction might contribute to other nuclear envelope-related disorders currently attributed to unknown mechanisms.